Mass spectrometry of steroidal compounds in multiplexed patient samples

ABSTRACT

The invention relates to the quantitative measurement of steroidal compounds by mass spectrometry. In a particular aspect, the invention relates to methods for quantitative measurement of steroidal compounds from multiple samples by mass spectrometry.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation application of U.S. application Ser. No. 15/362,210, filed Nov. 28, 2016, now U.S. Pat. No. 10,955,424, which is a continuation application of U.S. application Ser. No. 14/715,153, filed May 18, 2015, now U.S. Pat. No. 9,506,937, which is a continuation application of U.S. application Ser. No. 13/514,892, filed Jun. 8, 2012, now U.S. Pat. No. 9,034,653, which is a national stage application of International Application Number PCT/US2010/059746, filed Dec. 9, 2010, which claims the benefit of U.S. Provisional Application Ser. No. 61/258,991, filed Dec. 11, 2009, each of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the quantitative measurement of steroidal compounds by mass spectrometry. In a particular aspect, the invention relates to methods for quantitative measurement of steroidal compounds from multiple samples by mass spectrometry.

BACKGROUND OF THE INVENTION

Steroidal compounds are any of numerous naturally occurring or synthetic fat-soluble organic compounds having as a basis 17 carbon atoms arranged in four rings and including the sterols and bile acids, adrenal and sex hormones, certain natural drugs such as digitalis compounds, as well as certain vitamins and related compounds (such as vitamin D, vitamin D analogues, and vitamin D metabolites).

Many steroidal compounds are biologically important. For example, vitamin D is an essential nutrient with important physiological roles in the positive regulation of calcium (Ca²⁺) homeostasis. Vitamin D can be made de novo in the skin by exposure to sunlight or it can be absorbed from the diet. There are two forms of vitamin D; vitamin D₂ (ergocalciferol) and vitamin D₃ (cholecalciferol). Vitamin D₃ is the form synthesized de novo by animals. It is also a common supplement added to milk products and certain food products produced in the United States. Both dietary and intrinsically synthesized vitamin D₃ must undergo metabolic activation to generate the bioactive metabolites. In humans, the initial step of vitamin D₃ activation occurs primarily in the liver and involves hydroxylation to form the intermediate metabolite 25-hydroxycholecalciferol (calcifediol; 25OHD₃). Calcifediol is the major form of Vitamin D₃ in circulation. Circulating 25OHD₃ is then converted by the kidney to form 1.25-dihydroxyvitamin D₃ (calcitriol; 1.25(OH)₂D₃), which is generally believed to be the metabolite of Vitamin D₃ with the highest biological activity.

Vitamin D₂ is derived from fungal and plant sources. Many over-the-counter dietary supplements contain ergocalciferol (vitamin D₂) rather than cholecalciferol (vitamin D₃). Drisdol, the only high-potency prescription form of vitamin D available in the United States, is formulated with ergocalciferol. Vitamin D₂ undergoes a similar pathway of metabolic activation in humans as Vitamin D₃, forming the metabolites 25OHD₃ and 1.25(OH)₂D₂. Vitamin D₂ and vitamin D₃ have long been assumed to be biologically equivalent in humans, however recent reports suggest that there may be differences in the bioactivity and bioavailability of these two forms of vitamin D (Armas et. al., (2004) J. Clin. Endocrinol. Metab. 89:5387-5391).

Measurement of vitamin D, the inactive vitamin D precursor, is rare in clinical settings. Rather, serum levels of 25-hydroxyvitamin D₃, 25-hydroxyvitamin D₂, and total 25-hydroxyvitamin D (“25 OHD”) are useful indices of vitamin D nutritional status and the efficacy of certain vitamin D analogs. The measurement of 25 OHD is commonly used in the diagnosis and management of disorders of calcium metabolism. In this respect, low levels of 25 OHD are indicative of vitamin D deficiency associated with diseases such as hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, elevated alkaline phosphatase, osteomalacia in adults and rickets in children. In patients suspected of vitamin D intoxication, elevated levels of 25 OHD distinguishes this disorder from other disorders that cause hypercalcemia.

Measurement of 1.25(OH)₂D is also used in clinical settings. Certain disease states can be reflected by circulating levels of 1.25(OH)₂D, for example kidney disease and kidney failure often result in low levels of 1.25(OH)₂D. Elevated levels of 1.25(OH)₂D may be indicative of excess parathyroid hormone or can be indicative of certain diseases such as sarcoidosis or certain types of lymphomas.

Detection of vitamin D metabolites has been accomplished by radioimmunoassay with antibodies co-specific for 25OHD₂ and 25 OHD₃. Because the current immunologically-based assays do not separately resolve 25 OHD₂ and 25 OHD₃, the source of any nutritional deficiency of vitamin D cannot be determined without resorting to other tests. Reports have been published that disclose methods for detecting specific vitamin D metabolites using mass spectrometry. In some of the reports, the vitamin D metabolites are derivatized prior to mass spectrometry, but in others, they are not. For example Holmquist, et al., U.S. patent application Ser. No. 11/946,765, filed Dec. 28, 2007; Yeung B, et al., J Chromatogr. 1993, 645(1):115-23; Higashi T, et al., Steroids. 2000, 65(5):281-94; Higashi T, et al., Biol Pharm Bull. 2001, 24(7):738-43; Higashi T, et al., J Pharm Biomed Anal. 2002, 29(5):947-55; Higashi T, et al., Anal. Bioanal Chem, 2008, 391:229-38; and Aronov, et al., Anal Bioanal Chem, 2008, 391:1917-30 disclose methods for detecting various vitamin D metabolites by derivatizing the metabolites prior to mass spectrometry. Methods to detect underivatized vitamin D metabolites are reported in Clarke, et al., in U.S. patent application Ser. No. 11/101,166, filed Apr. 6, 2005, and Ser. No. 11/386,215, filed Mar. 21, 2006, and Singh, et al., in U.S. patent application Ser. No. 10/977,121, filed Oct. 24, 2004. Reports have also been published that disclose derivatization of vitamin D₃ with Cookson-type reagents, specifically 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) and 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQ-TAD). See Aberhart, J, et al., J. Org. Chem. 1976, 41(12):2098-2102, and Kamao, M, et al., J Chromatogr. B 2007, 859:192-200.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the amount of a steroidal compound in each of a plurality of test samples with a single mass spectrometric assay. The methods include processing each test sample differently to form a plurality of processed samples, wherein as a result of the processing, the steroidal compound in each processed sample is distinguishable by mass spectrometry from the steroidal compound in other processed samples; combining the processed samples to form a multiplex sample; subjecting the multiplex sample to an ionization source under conditions suitable to generate one or more ions detectable by mass spectrometry, wherein one or more ions generated from the steroidal compound from each processed sample are distinct from one or more ions from the steroidal compound from the other processed samples; detecting the amount of one or more ions from the steroidal compound from each processed sample by mass spectrometry; and relating the amount of one or more ions from the steroidal compound from each processed sample to the amount of the steroidal compound in each test sample.

In some embodiments, processing a test sample comprises subjecting each test sample to a different derivatizing agent under conditions suitable to generate derivatized steroidal compounds. In some embodiment, one test sample may be processed without subjecting the sample to a derivatizing agent.

In some embodiments, the different derivatizing agents used in the processing of the plurality of test samples are isotopic variants of each another. In some embodiments, the different derivatizing agents are Cookson-type derivatizing agents; such as Cookson-type derivatization agents selected from the group consisting of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQTAD), 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD), 4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD), and isotopic variants thereof. In one related embodiment, the Cookson-type derivatizing agents are isotopic variants of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). In a specific embodiment, the plurality of samples comprises two samples, a first Cookson-type derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), and a second Cookson-type derivatizing reagent is 13_(C) ₆ -4-phenyl-1,2,4-triazoline-3,5-dione (13_(C) ₆ -PTAD).

In some embodiments, the steroidal compound is a vitamin D or vitamin D related compound. In related embodiments, the steroidal compound is selected from the group consisting of vitamin D₂, vitamin D₃, 25-hydroxyvitamin D₂ (25OHD₂), 25-hydroxyvitamin D₃ (25OHD₃), 1α,25-dihydroxyvitamin D₂ (1α,25OHD₂), and 1α,25-dihydroxyvitamin D₃ (1α,25OHD₃). In a specific embodiment, the steroidal compound is 25-hydroxyvitamin D₂ (25OHD₂) or 25-hydroxyvitamin D₃ (25OHD₃).

The methods described above may be conducted for the analysis of two or more steroidal compounds in each of a plurality of test samples. In some of these embodiments, the two or more steroidal compounds in each test sample may include at least one steroidal compound selected from the group consisting of 25-hydroxyvitamin D₂ (25OHD₂) and 25-hydroxyvitamin D₃ (25OHD₃). In some embodiments, the two or more steroidal compounds in each test sample are 25-hydroxyvitamin D₂ (25OHD₂) and 25-hydroxyvitamin D₃ (25OHD₃).

In specific embodiments, the amount of one or more vitamin D or vitamin D related compounds in each of two test samples is determined with a single mass spectrometric assay. In this embodiment, a first processed sample is generated by subjecting a first test sample to a first isotopic variant of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) under conditions suitable to generate one or more vitamin D or vitamin D related derivatives; a second processed sample is generated by subjecting a second test sample to a second isotopic variant of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) under conditions suitable to generate one or more vitamin D or vitamin D related derivatives, wherein the first and second isotopic variant of PTAD are distinguishable by mass spectrometry; the first processed sample is mixed with the second processed sample to form a multiplex sample; one or more vitamin D or vitamin D related derivatives from each processed sample in the multiplex sample are subjected to an ionization source under conditions suitable to generate one or more ions detectable by mass spectrometry, wherein one or more ions from each vitamin D or vitamin D related derivatives from the first processed sample are distinct from the one or more ions from vitamin D or vitamin D related derivatives from the second processed sample; the amounts of one or more ions from one or more vitamin D or vitamin D related derivatives from each processed sample are determined by mass spectrometry; and the amounts of the determined ions are related to the amounts of vitamin D or vitamin D related compound in the first or second test sample.

In some specific embodiments, the first isotopic variant of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), and the second isotopic variant of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) is 13_(C) ₆ -4-phenyl-1,2,4-triazoline-3,5-dione (13_(C) ₆ -PTAD).

In some specific embodiments, the one or more vitamin D or vitamin D related compounds are selected from the group consisting of 25-hydroxyvitamin D₂ (25OHD₂) and 25-hydroxyvitamin D₃ (25OHD₃). In some related specific embodiments, the one or more vitamin D or vitamin D related compounds include 25-hydroxyvitamin D₂ (25OHD₂) and 25-hydroxyvitamin D₃ (25OHD₃). In some related specific embodiments, the one or more vitamin D or vitamin D related compounds are 25-hydroxyvitamin D₂ (25OHD₂) and 25-hydroxyvitamin D₃ (25OHD₃).

In some embodiments, the multiplex sample is subjected to an extraction column and an analytical column prior to being subjected to an ionization source. In some related embodiments, the extraction column is a solid-phase extraction (SPE) column. In other related embodiments, the extraction column is a turbulent flow liquid chromatography (TFLC) column. In some embodiments, the analytical column is a high performance liquid chromatography (HPLC) column.

In embodiments which utilize two or more of an extraction column, an analytical column, and an ionization source, two or more of these components may be connected in an on-line fashion to allow for automated sample processing and analysis.

In the methods described herein, mass spectrometry may be tandem mass spectrometry. In embodiments utilizing tandem mass spectrometry, tandem mass spectrometry may be conducted by any method known in the art, including for example, multiple reaction monitoring, precursor ion scanning, or product ion scanning.

In the methods described herein, steroidal compounds may be ionized by any suitable ionization technique known in the art. In some embodiments, the ionization source is a laser diode thermal desorption (LDTD) ionization source.

In preferred embodiments, the test samples comprise biological samples, such as plasma or serum.

As used herein, the term “multiplex sample” refers to a sample prepared by pooling two or more samples to form the single “multiplex” sample which is then subject to mass spectrometric analysis. In the methods described herein, two or more test samples are each processed differently to generate multiple differently processed samples. These multiple differently processed samples are then pooled to generate a single “multiplex” sample, which is then subject to mass spectrometric analysis.

As used herein, the term “steroidal compound” refers to any of numerous naturally occurring or synthetic fat-soluble organic compounds having as a basis 17 carbon atoms arranged in four rings and including the sterols and bile acids, adrenal and sex hormones, certain natural drugs such as digitalis compounds, as well as certain vitamins and related compounds (such as vitamin D, vitamin D analogues, and vitamin D metabolites).

As used herein, the term “vitamin D or vitamin D related compound” refers to any natural or synthetic form of vitamin D, or any chemical species related to vitamin D generated by a transformation of vitamin D, such as intermediates and products of vitamin D metabolism. For example, vitamin D may refer to one or more of vitamin D₂ and vitamin D₃. Vitamin D may also be referred to as “nutritional” vitamin D to distinguish from chemical species generated by a transformation of vitamin D. Vitamin D related compounds may include chemical species generated by biotransformation of analogs of, or a chemical species related to, vitamin D₂ or vitamin D₃. Vitamin D related compounds, specifically vitamin D metabolites, may be found in the circulation of an animal and/or may be generated by a biological organism, such as an animal. Vitamin D metabolites may be metabolites of naturally occurring forms of vitamin D or may be metabolites of synthetic vitamin D analogs. In certain embodiments, vitamin D related compounds may include one or more vitamin D metabolites selected from the group consisting of 25-hydroxyvitamin D₃, 25-hydroxyvitamin D₂, 1α,25-dihydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₂.

As used herein, “derivatizing” means reacting two molecules to form a new molecule. Thus, a derivatizing agent is an agent that is reacted with another substance to derivatize the substance. For example, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) is a derivatizing reagent that may be reacted with a vitamin D metabolite to form a PTAD-derivatized vitamin D metabolite.

As used herein, “different derivatizing agents” are derivatizing agents that are distinguishable by mass spectrometry. As one example, two isotopic variants of the same derivatizing agent are distinguishable by mass spectrometry. As another example, halogenated variants of the same derivatizing agent are also distinguishable by mass spectrometry. For example, halogenated and non-halogenated versions of the same Cookson-type agent, such as 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), may be used. Further, two halogenated versions of the same Cookson-type agent, but halogenated with different halogens or with different numbers of halogens, may be used. As another example, two different Cookson-type agents, such as 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), 4-methyl-1,2,4-triazoline-3,5-dione (MTAD), and 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD), may be used. The above examples illustrate the principle of derivatizing agents that are distinguishable by mass spectrometry. Other examples, including combinations of any of the above, may be possible as would be appreciated by one of skill in the art.

As used herein, the names of derivatized forms of steroidal compounds include an indication as to the nature of derivatization. For example, the PTAD derivative of 25-hydroxyvitamin D₂ is indicated as PTAD-25-hydroxyvitamin D₂ (or PTAD-25OHD₂).

As used herein, a “Cookson-type derivatizing agent” is a 4-substituted 1,2,4-triazoline-3,5-dione compound. Exemplary Cookson-type derivatizing agents include 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), 4-methyl-1,2,4-triazoline-3,5-dione (MTAD), 4-[2-6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQTAD), 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD), and 4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD). Additionally, isotopically labeled variants of Cookson-type derivatizing agents may be used in some embodiments. For example, the 13_(C) ₆ -PTAD isotopic variant is 6 mass units heavier than normal PTAD and may be used in some embodiments. Derivatization of steroidal compounds, including vitamin D and vitamin D related compounds, by Cookson-type reagents can be conducted by any appropriate method. See, e.g., Holmquist, et al., U.S. patent application Ser. No. 11/946,765, filed Dec. 28, 2007; Yeung B, et al., J Chromatogr. 1993, 645(1):115-23; Higashi T, et al., Steroids. 2000, 65(5):281-94; Higashi T, et al., Biol Pharm Bull. 2001, 24(7):738-43; Higashi T, et al., J Pharm Biomed Anal. 2002, 29(5):947-55; Higashi T, et al., Anal. Biochanal Chem, 2008, 391:229-38; and Aronov, et al., Anal Bioanal Chem, 2008, 391:1917-30.

In certain preferred embodiments of the methods disclosed herein, mass spectrometry is performed in positive ion mode. Alternatively, mass spectrometry is performed in negative ion mode. Various ionization sources, including for example atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI), may be used in embodiments of the present invention. In certain embodiments, steroidal compounds, including vitamin D and vitamin D related compounds, are measured using APCI in positive ion mode.

In preferred embodiments, one or more separately detectable internal standards are provided in the sample, the amount of which are also determined in the sample. In these embodiments, all or a portion of both the analyte(s) of interest and the internal standard(s) present in the sample are ionized to produce a plurality of ions detectable in a mass spectrometer, and one or more ions produced from each are detected by mass spectrometry. Exemplary internal standard(s) include vitamin D₂-[6, 19, 19]-2_(H) ₃ , vitamin D₂-[24, 24, 24, 25, 25, 25]-2_(H) ₆ , vitamin D₃-[6, 19, 19]-2_(H) ₃ , vitamin D₃-[24, 24, 24, 25, 25, 25]-2_(H) ₆ , 25OHD₂-[6, 19, 19]-2_(H) ₃ , 25OHD₂-[24, 24, 24, 25, 25, 25]-2_(H) ₆ , 25OHD₃-[6, 19, 19]-2_(H) ₃ , 25OHD₃-[24, 24, 24, 25, 25, 25]-2_(H) ₆ , 1α,25OHD₂-[6, 19, 19]-2_(H) ₃ , 1α,25OHD₂-[24, 24, 24, 25, 25, 25]-2_(H) ₆ , 1α,25OHD₃-[6, 19, 19]-2_(H) ₃ , 1α,25OHD₃-[24, 24, 24, 25, 25, 25]-2_(H) ₆ .

One or more separately detectable internal standards may be provided in the sample prior to treatment of the sample with a Cookson-type derivatizing reagent. In these embodiments, the one or more internal standards may undergo derivatization along with the endogenous steroidal compounds, in which case ions of the derivatized internal standards are detected by mass spectrometry. In these embodiments, the presence or amount of ions generated from the analyte of interest may be related to the presence of amount of analyte of interest in the sample. In some embodiments, the internal standards may be isotopically labeled versions of steroidal compounds under investigation. For example in an assay where vitamin D metabolites are analytes of interest, 25OHD₂-[6, 19, 19]-2_(H) ₃ or 25OHD₃-[6, 19, 19]-2_(H) ₃ may be used as an internal standard. In embodiments where 25OHD₂-[6, 19, 19]-2_(H) ₃ is used as internal standards, PTAD-25OHD₂-[6, 19, 19]-2_(H) ₃ ions detectable in a mass spectrometer are selected from the group consisting of positive ions with a mass/charge ratio (m/z) of 573.30±0.50 and 301.10±0.50. In related embodiments, a PTAD-25OHD₂-[6, 19, 19]-2_(H) ₃ precursor ion has a m/z of 573.30±0.50, and a fragment ion has m/z of 301.10±0.50. In embodiments where 25OHD₃-[6, 19, 19]-2_(H) ₃ is used as an internal standard, PTAD-25OHD₃-[6, 19, 19] ions detectable in a mass spectrometer are selected from the group consisting of positive ions with a mass/charge ratio (m/z) of 561.30±0.50 and 301.10±0.50. In related embodiments, a PTAD-25OHD₃-[6, 19, 19] precursor ion has a m/z of 561.30±0.50, and a fragment ion has m/z of 301.10±0.50.

As used herein, an “isotopic label” produces a mass shift in the labeled molecule relative to the unlabeled molecule when analyzed by mass spectrometric techniques. Examples of suitable labels include deuterium (2_(H)), 13_(C), and 15_(N). For example, 25OHD₂-[6, 19, 19] and 25OHD₃-[6, 19, 19] have masses about 3 mass units higher than 25OHD₂ and 25OHD₃. The isotopic label can be incorporated at one or more positions in the molecule and one or more kinds of isotopic labels can be used on the same isotopically labeled molecule.

In other embodiments, the amount of the vitamin D metabolite ion or ions may be determined by comparison to one or more external reference standards. Exemplary external reference standards include blank plasma or serum spiked with one or more of 25OHD₂, 25OHD₂-[6, 19, 19], 25OHD₃, and 25OHD₃-[6, 19, 19]. External standards typically will undergo the same treatment and analysis as any other sample to be analyzed, including treatment with one or more Cookson-type reagents prior to mass spectrometry.

In certain preferred embodiments, the limit of quantitation (LOQ) of 25OHD₂ is within the range of 1.9 ng/mL to 10 ng/mL, inclusive; preferably within the range of 1.9 ng/mL to 5 ng/mL, inclusive; preferably about 1.9 ng/mL. In certain preferred embodiments, the limit of quantitation (LOQ) of 25OHD₃ is within the range of 3.3 ng/mL to 10 ng/mL, inclusive; preferably within the range of 3.3 ng/mL to 5 ng/mL, inclusive; preferably about 3.3 ng/mL.

As used herein, unless otherwise stated, the singular forms “a,” “an,” and “the” include plural reference. Thus, for example, a reference to “a protein” includes a plurality of protein molecules.

As used herein, the term “purification” or “purifying” does not refer to removing all materials from the sample other than the analyte(s) of interest. Instead, purification refers to a procedure that enriches the amount of one or more analytes of interest relative to other components in the sample that may interfere with detection of the analyte of interest. Purification of the sample by various means may allow relative reduction of one or more interfering substances, e.g., one or more substances that may or may not interfere with the detection of selected parent or daughter ions by mass spectrometry. Relative reduction as this term is used does not require that any substance, present with the analyte of interest in the material to be purified, is entirely removed by purification.

As used herein, the term “solid phase extraction” or “SPE” refers to a process in which a chemical mixture is separated into components as a result of the affinity of components dissolved or suspended in a solution (i.e., mobile phase) for a solid through or around which the solution is passed (i.e., solid phase). In some instances, as the mobile phase passes through or around the solid phase, undesired components of the mobile phase may be retained by the solid phase resulting in a purification of the analyte in the mobile phase. In other instances, the analyte may be retained by the solid phase, allowing undesired components of the mobile phase to pass through or around the solid phase. In these instances, a second mobile phase is then used to elute the retained analyte off of the solid phase for further processing or analysis. SPE, including TFLC, may operate via a unitary or mixed mode mechanism. Mixed mode mechanisms utilize ion exchange and hydrophobic retention in the same column; for example, the solid phase of a mixed-mode SPE column may exhibit strong anion exchange and hydrophobic retention; or may exhibit column exhibit strong cation exchange and hydrophobic retention.

As used herein, the term “chromatography” refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s). Examples of “liquid chromatography” include reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), and turbulent flow liquid chromatography (TFLC) (sometimes known as high turbulence liquid chromatography (HTLC) or high throughput liquid chromatography).

As used herein, the term “high performance liquid chromatography” or “HPLC” (sometimes known as “high pressure liquid chromatography”) refers to liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column.

As used herein, the term “turbulent flow liquid chromatography” or “TFLC” (sometimes known as high turbulence liquid chromatography or high throughput liquid chromatography) refers to a form of chromatography that utilizes turbulent flow of the material being assayed through the column packing as the basis for performing the separation. TFLC has been applied in the preparation of samples containing two unnamed drugs prior to analysis by mass spectrometry. See, e.g., Zimmer et al., J Chromatogr A 854: 23-35 (1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368, 5,795,469, and 5,772,874, which further explain TFLC. Persons of ordinary skill in the art understand “turbulent flow”. When fluid flows slowly and smoothly, the flow is called “laminar flow”. For example, fluid moving through an HPLC column at low flow rates is laminar. In laminar flow the motion of the particles of fluid is orderly with particles moving generally in straight lines. At faster velocities, the inertia of the water overcomes fluid frictional forces and turbulent flow results. Fluid not in contact with the irregular boundary “outruns” that which is slowed by friction or deflected by an uneven surface. When a fluid is flowing turbulently, it flows in eddies and whirls (or vortices), with more “drag” than when the flow is laminar. Many references are available for assisting in determining when fluid flow is laminar or turbulent (e.g., Turbulent Flow Analysis: Measurement and Prediction, P. S. Bernard & J. M. Wallace, John Wiley & Sons, Inc., (2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers to chromatography in which the sample mixture is vaporized and injected into a stream of carrier gas (as nitrogen or helium) moving through a column containing a stationary phase composed of a liquid or a particulate solid and is separated into its component compounds according to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column” refers to a chromatography column containing an average particle diameter greater than about 50 μm.

As used herein, the term “analytical column” refers to a chromatography column having sufficient chromatographic plates to effect a separation of materials in a sample that elute from the column sufficient to allow a determination of the presence or amount of an analyte. In a preferred embodiment the analytical column contains particles of about 5 μm in diameter. Such columns are often distinguished from “extraction columns”, which have the general purpose of separating or extracting retained material from non-retained materials in order to obtain a purified sample for further analysis.

As used herein, the terms “on-line” and “inline”, for example as used in “on-line automated fashion” or “on-line extraction” refers to a procedure performed without the need for operator intervention. In contrast, the term “off-line” as used herein refers to a procedure requiring manual intervention of an operator. Thus, if samples are subjected to precipitation, and the supernatants are then manually loaded into an autosampler, the precipitation and loading steps are off-line from the subsequent steps. In various embodiments of the methods, one or more steps may be performed in an on-line automated fashion.

As used herein, the term “mass spectrometry” or “MS” refers to an analytical technique to identify compounds by their mass. MS refers to methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or “m/z”. MS technology generally includes (1) ionizing the compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating a mass-to-charge ratio. The compounds may be ionized and detected by any suitable means. A “mass spectrometer” generally includes an ionizer and an ion detector. In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrometric instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,204,500, entitled “Mass Spectrometry From Surfaces;” U.S. Pat. No. 6,107,623, entitled “Methods and Apparatus for Tandem Mass Spectrometry;” U.S. Pat. No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry;” U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes;” Wright et al., Prostate Cancer and Prostatic Diseases 1999, 2: 264-76; and Merchant and Weinberger, Electrophoresis 2000, 21: 1164-67.

As used herein, the term “operating in negative ion mode” refers to those mass spectrometry methods where negative ions are generated and detected. The term “operating in positive ion mode” as used herein, refers to those mass spectrometry methods where positive ions are generated and detected.

As used herein, the term “ionization” or “ionizing” refers to the process of generating an analyte ion having a net electrical charge equal to one or more electron units. Negative ions are those having a net negative charge of one or more electron units, while positive ions are those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methods in which an analyte of interest in a gaseous or vapor phase interacts with a flow of electrons. Impact of the electrons with the analyte produces analyte ions, which may then be subjected to a mass spectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methods in which a reagent gas (e.g. ammonia) is subjected to electron impact, and analyte ions are formed by the interaction of reagent gas ions and analyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers to methods in which a beam of high energy atoms (often Xe or Ar) impacts a non-volatile sample, desorbing and ionizing molecules contained in the sample. Test samples are dissolved in a viscous liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether, 2-nitrophenyloctyl ether, sulfolane, diethanolamine, and triethanolamine. The choice of an appropriate matrix for a compound or sample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization” or “MALDI” refers to methods in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photo-ionization, protonation, deprotonation, and cluster decay. For MALDI, the sample is mixed with an energy-absorbing matrix, which facilitates desorption of analyte molecules.

As used herein, the term “surface enhanced laser desorption ionization” or “SELDI” refers to another method in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photo-ionization, protonation, deprotonation, and cluster decay. For SELDI, the sample is typically bound to a surface that preferentially retains one or more analytes of interest. As in MALDI, this process may also employ an energy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers to methods in which a solution is passed along a short length of capillary tube, to the end of which is applied a high positive or negative electric potential. Solution reaching the end of the tube is vaporized (nebulized) into a jet or spray of very small droplets of solution in solvent vapor. This mist of droplets flows through an evaporation chamber, which is heated slightly to prevent condensation and to evaporate solvent. As the droplets get smaller the electrical surface charge density increases until such time that the natural repulsion between like charges causes ions as well as neutral molecules to be released.

As used herein, the term “atmospheric pressure chemical ionization” or “APCI,” refers to mass spectrometry methods that are similar to ESI; however, APCI produces ions by ion-molecule reactions that occur within a plasma at atmospheric pressure. The plasma is maintained by an electric discharge between the spray capillary and a counter electrode. Then ions are typically extracted into the mass analyzer by use of a set of differentially pumped skimmer stages. A counterflow of dry and preheated N₂ gas may be used to improve removal of solvent. The gas-phase ionization in APCI can be more effective than ESI for analyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used herein refers to the form of mass spectrometry where the mechanism for the photoionization of molecule M is photon absorption and electron ejection to form the molecular ion M+. Because the photon energy typically is just above the ionization potential, the molecular ion is less susceptible to dissociation. In many cases it may be possible to analyze samples without the need for chromatography, thus saving significant time and expense. In the presence of water vapor or protic solvents, the molecular ion can extract H to form MH+. This tends to occur if M has a high proton affinity. This does not affect quantitation accuracy because the sum of M+ and MH+ is constant. Drug compounds in protic solvents are usually observed as MH+, whereas nonpolar compounds such as naphthalene or testosterone usually form M+. See, e.g., Robb et al., Anal. Chem. 2000, 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers to methods in which a sample interacts with a partially ionized gas at a sufficiently high temperature such that most elements are atomized and ionized.

As used herein, the term “field desorption” refers to methods in which a non-volatile test sample is placed on an ionization surface, and an intense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of an analyte from a surface and/or the entry of an analyte into a gaseous phase. Laser diode thermal desorption (LDTD) is a technique wherein a sample containing the analyte is thermally desorbed into the gas phase by a laser pulse. The laser hits the back of a specially made 96-well plate with a metal base. The laser pulse heats the base and the heat causes the sample to transfer into the gas phase. The gas phase sample is then drawn into an ionization source, where the gas phase sample is ionized in preparation for analysis in the mass spectrometer. When using LDTD, ionization of the gas phase sample may be accomplished by any suitable technique known in the art, such as by ionization with a corona discharge (for example by APCI).

As used herein, the term “selective ion monitoring” is a detection mode for a mass spectrometric instrument in which only ions within a relatively narrow mass range, typically about one mass unit, are detected.

As used herein, “multiple reaction mode,” sometimes known as “selected reaction monitoring,” is a detection mode for a mass spectrometric instrument in which a precursor ion and one or more fragment ions are selectively detected.

As used herein, the term “lower limit of quantification”, “lower limit of quantitation” or “LLOQ” refers to the point where measurements become quantitatively meaningful. The analyte response at this LOQ is identifiable, discrete and reproducible with a relative standard deviation (RSD %) of less than 20% and an accuracy of 80% to 120%.

As used herein, the term “limit of detection” or “LOD” is the point at which the measured value is larger than the uncertainty associated with it. The LOD is the point at which a value is beyond the uncertainty associated with its measurement and is defined as three times the RSD of the mean at the zero concentration.

As used herein, an “amount” of an analyte in a body fluid sample refers generally to an absolute value reflecting the mass of the analyte detectable in volume of sample. However, an amount also contemplates a relative amount in comparison to another analyte amount. For example, an amount of an analyte in a sample can be an amount which is greater than a control or normal level of the analyte normally present in the sample.

The term “about” as used herein in reference to quantitative measurements not including the measurement of the mass of an ion, refers to the indicated value plus or minus 10%. Mass spectrometry instruments can vary slightly in determining the mass of a given analyte. The term “about” in the context of the mass of an ion or the mass/charge ratio of an ion refers to +/−0.50 atomic mass unit.

The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an exemplary chromatogram for PTAD-25OHD₃. FIG. 1B shows an exemplary chromatogram for PTAD-25OHD₃-[6, 19, 19]-2_(H) ₃ (internal standard). FIG. 1C shows an exemplary chromatogram for PTAD-25OHD₂. FIG. 1D shows an exemplary chromatogram for PTAD-25OHD₂-[6, 19, 19]-2_(H) ₃ (internal standard). Details are discussed in Example 3.

FIGS. 2A and 2B show exemplary calibration curves for 25OHD₂ and 25OHD₃ in serum samples determined by methods described in Example 3.

FIG. 3A shows a plots of coefficient of variation versus concentration for 25OHD₂ and 25OHD₃. FIG. 3B shows the same plot expanded near the LLOQ. Details are described in Example 4.

FIGS. 4A-B show linear regression and Deming regression analyses for the comparison of mass spectrometric determination of 25OHD₂ with and without PTAD derivatization. Details are described in Example 10.

FIGS. 5A-B show linear regression and Deming regression analyses for the comparison of mass spectrometric determination of 25OHD₃ with and without PTAD derivatization. Details are described in Example 10.

FIGS. 6A-D show plots comparing the results of analysis of multiplex samples and unmixed samples (with the same derivatization agent). Details are described in Example 14.

FIGS. 7A-D are plots comparing the results of analysis of the same specimen treated with different derivatization agents (but comparing mixed versus mixed, or unmixed versus unmixed samples). Details are described in Example 14.

FIGS. 8A-D are plots comparing the results of analysis of the same specimen treated with different derivatization agents, with one analysis coming from a mixed sample and one coming from an unmixed sample. Details are described in Example 14.

FIG. 9A shows an exemplary Q1 scan spectrum (covering the m/z range of about 350 to 450) for 25-hydroxyvitamin D₂ ions. FIG. 9B shows an exemplary product ion spectra (covering the m/z range of about 100 to 396) for fragmentation of the 25-hydroxyvitamin D₂ precursor ion with m/z of about 395.2. Details are described in Example 15.

FIG. 10A shows an exemplary Q1 scan spectrum (covering the m/z range of about 350 to 450) for 25-hydroxyvitamin D₃ ions. FIG. 10B shows an exemplary product ion spectra (covering the m/z range of about 100 to 396) for fragmentation of the 25-hydroxyvitamin D₃ precursor ion with m/z of about 383.2. Details are described in Example 15.

FIG. 11A shows an exemplary Q1 scan spectrum (covering the m/z range of about 520 to 620) for PTAD-25-hydroxyvitamin D₂ ions. FIG. 11B shows an exemplary product ion spectra (covering the m/z range of about 200 to 400) for fragmentation of the PTAD-25-hydroxyvitamin D₂ precursor ion with m/z of about 570.3. Details are described in Example 15.

FIG. 12A shows an exemplary Q1 scan spectrum (covering the m/z range of about 520 to 620) for PTAD-25-hydroxyvitamin D₃ ions. FIG. 12B shows an exemplary product ion spectra (covering the m/z range of about 200 to 400) for fragmentation of the PTAD-25-hydroxyvitamin D₃ precursor ion with m/z of about 558.3. Details are described in Example 15.

FIG. 13A shows an exemplary Q1 scan spectrum (covering the m/z range of about 520 to 620) for PTAD-1α,25-dihydroxyvitamin D₂ ions. FIG. 13B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-1α,25-dihydroxyvitamin D₂ precursor ion with m/z of about 550.4. FIG. 13C shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-1α,25-dihydroxyvitamin D₂ precursor ion with m/z of about 568.4. FIG. 13D shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-1α,25-dihydroxyvitamin D₂ precursor ion with m/z of about 586.4. Details are described in Example 16.

FIG. 14A shows an exemplary Q1 scan spectrum (covering the m/z range of about 520 to 620) for PTAD-1α,25-hydroxyvitamin D₃ ions. FIG. 14B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-1α,25-dihydroxyvitamin D₃-PTAD precursor ion with m/z of about 538.4. FIG. 14C shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-1α,25-dihydroxyvitamin D₃ precursor ion with m/z of about 556.4. FIG. 14D shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-1α,25-dihydroxyvitamin D₃ precursor ion with m/z of about 574.4. Details are described in Example 16.

FIG. 15A shows an exemplary Q1 scan spectrum (covering the m/z range of about 500 to 620) for PTAD-vitamin D₂ ions. FIG. 15B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-vitamin D₂ precursor ion with m/z of about 572.2. Details are described in Example 17.

FIG. 16A shows an exemplary Q1 scan spectrum (covering the m/z range of about 500 to 620) for PTAD-vitamin D₃ ions. FIG. 16B shows an exemplary product ion spectra (covering the m/z range of about 250 to 350) for fragmentation of the PTAD-vitamin D₃ precursor ion with m/z of about 560.2. Details are described in Example 17.

DETAILED DESCRIPTION OF THE INVENTION

Methods are described for measuring steroidal compounds, such as vitamin D and vitamin D related compounds, in a sample. More specifically, methods are described for detecting and quantifying steroidal compounds in a plurality of test samples in a single mass spectrometric assay. The methods may utilize Cookson-type reagents, such as PTAD, to generate derivatized steroidal compounds combined with methods of mass spectrometry (MS), thereby providing a high-throughput assay system for detecting and quantifying steroidal compounds in a plurality of test samples. The preferred embodiments are particularly well suited for application in large clinical laboratories for automated steroidal compound quantification.

Suitable test samples for use in methods of the present invention include any test sample that may contain the analyte of interest. In some preferred embodiments, a sample is a biological sample; that is, a sample obtained from any biological source, such as an animal, a cell culture, an organ culture, etc. In certain preferred embodiments, samples are obtained from a mammalian animal, such as a dog, cat, horse, etc. Particularly preferred mammalian animals are primates, most preferably male or female humans. Preferred samples comprise bodily fluids such as blood, plasma, serum, saliva, cerebrospinal fluid, or tissue samples; preferably plasma (including EDTA and heparin plasma) and serum; most preferably serum. Such samples may be obtained, for example, from a patient; that is, a living person, male or female, presenting oneself in a clinical setting for diagnosis, prognosis, or treatment of a disease or condition.

The present invention also contemplates kits for quantitation of one or more steroidal compounds. A kit for a steroidal compound quantitation assay may include a kit comprising the compositions provided herein. For example, a kit may include packaging material and measured amounts of an isotopically labeled internal standard, in amounts sufficient for at least one assay. Typically, the kits will also include instructions recorded in a tangible form (e.g., contained on paper or an electronic medium) for using the packaged reagents for use in a steroidal compound quantitation assay.

Calibration and QC pools for use in embodiments of the present invention are preferably prepared using a matrix similar to the intended sample matrix.

Sample Preparation for Mass Spectrometric Analysis

In preparation for mass spectrometric analysis, one or more steroidal compounds may be enriched relative to one or more other components in the sample (e.g. protein) by various methods known in the art, including for example, liquid chromatography, filtration, centrifugation, thin layer chromatography (TLC), electrophoresis including capillary electrophoresis, affinity separations including immunoaffinity separations, extraction methods including ethyl acetate or methanol extraction, and the use of chaotropic agents or any combination of the above or the like. These enrichment steps may be applied to individual test samples prior to processing, individual processed samples after derivatization, or to a multiplex sample after processed samples have been combined.

Protein precipitation is one method of preparing a sample, especially a biological sample, such as serum or plasma. Protein purification methods are well known in the art, for example, Polson et al., Journal of Chromatography B 2003, 785:263-275, describes protein precipitation techniques suitable for use in methods of the present invention. Protein precipitation may be used to remove most of the protein from the sample leaving one or more steroidal compounds in the supernatant. The samples may be centrifuged to separate the liquid supernatant from the precipitated proteins; alternatively the samples may be filtered to remove precipitated proteins. The resultant supernatant or filtrate may then be applied directly to mass spectrometry analysis; or alternatively to liquid chromatography and subsequent mass spectrometry analysis. In certain embodiments, individual test samples, such as plasma or serum, may be purified by a hybrid protein precipitation/liquid-liquid extraction method. In these embodiments, an unprocessed test sample is mixed with methanol, ethyl acetate, and water, and the resulting mixture is vortexed and centrifuged. The resulting supernatant, containing one or more purified steroidal compounds, is removed, dried to completion and reconstituted in acetonitrile. The one or more purified steroidal compounds in the acetonitrile solution may then be derivatized with any Cookson-type reagent, preferably PTAD or an isotopically labeled variant thereof.

Another method of sample purification that may be used prior to mass spectrometry is liquid chromatography (LC). Certain methods of liquid chromatography, including HPLC, rely on relatively slow, laminar flow technology. Traditional HPLC analysis relies on column packing in which laminar flow of the sample through the column is the basis for separation of the analyte of interest from the sample. The skilled artisan will understand that separation in such columns is a diffusional process and may select LC, including HPLC, instruments and columns that are suitable for use with derivatized steroidal compounds. The chromatographic column typically includes a medium (i.e., a packing material) to facilitate separation of chemical moieties (i.e., fractionation). The medium may include minute particles, or may include a monolithic material with porous channels. A surface of the medium typically includes a bonded surface that interacts with the various chemical moieties to facilitate separation of the chemical moieties. One suitable bonded surface is a hydrophobic bonded surface such as an alkyl bonded, cyano bonded surface, or highly pure silica surface. Alkyl bonded surfaces may include C-4, C-8, C-12, or C-18 bonded alkyl groups. In preferred embodiments, the column is a highly pure silica column (such as a Thermo Hypersil Gold Aq column). The chromatographic column includes an inlet port for receiving a sample and an outlet port for discharging an effluent that includes the fractionated sample. The sample may be supplied to the inlet port directly, or from an extraction column, such as an on-line SPE cartridge or a TFLC extraction column. In preferred embodiments, a multiplex sample may be purified by liquid chromatography prior to mass spectrometry.

In one embodiment, the multiplex sample may be applied to the LC column at the inlet port, eluted with a solvent or solvent mixture, and discharged at the outlet port. Different solvent modes may be selected for eluting the analyte(s) of interest. For example, liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polytyptic (i.e. mixed) mode. During chromatography, the separation of materials is effected by variables such as choice of eluent (also known as a “mobile phase”), elution mode, gradient conditions, temperature, etc.

In certain embodiments, analytes may be purified by applying a multiplex sample to a column under conditions where analytes of interest are reversibly retained by the column packing material, while one or more other materials are not retained. In these embodiments, a first mobile phase condition can be employed where the analytes of interest are retained by the column, and a second mobile phase condition can subsequently be employed to remove retained material from the column once the non-retained materials are washed through. Alternatively, analytes may be purified by applying a multiplex sample to a column under mobile phase conditions where the analytes of interest elute at a differential rates in comparison to one or more other materials. Such procedures may enrich the amount of an analyte of interest in the eluent at a particular time (i.e, a characteristic retention time) relative to one or more other components of the sample.

In one preferred embodiment, HPLC is conducted with an alkyl bonded analytical column chromatographic system. In certain preferred embodiments, a highly pure silica column (such as a Thermo Hypersil Gold Aq column) is used. In certain preferred embodiments, HPLC and/or TFLC are performed using HPLC Grade water as mobile phase A and HPLC Grade ethanol as mobile phase B.

By careful selection of valves and connector plumbing, two or more chromatography columns may be connected as needed such that material is passed from one to the next without the need for any manual steps. In preferred embodiments, the selection of valves and plumbing is controlled by a computer pre-programmed to perform the necessary steps. Most preferably, the chromatography system is also connected in such an on-line fashion to the detector system, e.g., an MS system. Thus, an operator may place a tray of samples in an autosampler, and the remaining operations are performed under computer control, resulting in purification and analysis of all samples selected.

In some embodiments, an extraction column may be used for purification of steroidal compounds prior to mass spectrometry. In such embodiments, samples may be extracted using a extraction column which captures the analyte, then eluted and chromatographed on a second extraction column or on an analytical HPLC column prior to ionization. For example, sample extraction with a TFLC extraction column may be accomplished with a large particle size (50 μm) packed column. Sample eluted off of this column may then be transferred to an HPLC analytical column for further purification prior to mass spectrometry. Because the steps involved in these chromatography procedures may be linked in an automated fashion, the requirement for operator involvement during the purification of the analyte can be minimized. This feature may result in savings of time and costs, and eliminate the opportunity for operator error.

In some embodiments, protein precipitation is accomplished with a hybrid protein precipitation/liquid-liquid extraction method which includes methanol protein precipitation and ethyl acetate/water extraction from serum test samples. The resulting steroidal compounds may be derivatized prior to being subjected to an extraction column. Preferably, the hybrid protein precipitation/liquid-liquid extraction method and the extraction column are connected in an on-line fashion. In preferred embodiments where the steroidal compounds are selected from the group consisting of vitamin D and vitamin D related compounds, the extraction column is preferably a C-8 extraction column, such as a Cohesive Technologies C8XL online extraction column (50 μm particle size, 0.5×50 mm) or equivalent. The eluent from the extraction column may then be applied to an analytical LC column, such as a HPLC column in an on-line fashion, prior to mass spectrometric analysis. Because the steps involved in these chromatography procedures may be linked in an automated fashion, the requirement for operator involvement during the purification of the analyte can be minimized. This feature may result in savings of time and costs, and eliminate the opportunity for operator error.

Detection and Quantitation by Mass Spectrometry

In various embodiments, derivatized steroidal compounds may be ionized by any method known to the skilled artisan. Mass spectrometry is performed using a mass spectrometer, which includes an ion source for ionizing the fractionated sample and creating charged molecules for further analysis. For example ionization of the sample may be performed by electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), fast atom bombardment (FAB), liquid secondary ionization (LSI), matrix assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, surface enhanced laser desorption ionization (SELDI), inductively coupled plasma (ICP), particle beam ionization, and LDTD. The skilled artisan will understand that the choice of ionization method may be determined based on the analyte to be measured, type of sample, the type of detector, the choice of positive versus negative mode, etc.

Derivatized steroidal compounds may be ionized in positive or negative mode. In preferred embodiments, derivatized steroidal compounds are ionized by APCI in positive mode. In related preferred embodiments, derivatized steroidal compounds ions are in a gaseous state and the inert collision gas is argon or nitrogen; preferably argon.

In mass spectrometry techniques generally, after the sample has been ionized, the positively or negatively charged ions thereby created may be analyzed to determine a mass-to-charge ratio. Suitable analyzers for determining mass-to-charge ratios include quadrupole analyzers, ion traps analyzers, and time-of-flight analyzers. Exemplary ion trap methods are described in Bartolucci, et al., Rapid Commun. Mass Spectrom. 2000, 14:967-73.

The ions may be detected using several detection modes. For example, selected ions may be detected, i.e. using a selective ion monitoring mode (SIM), or alternatively, mass transitions resulting from collision induced dissociation or neutral loss may be monitored, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). Preferably, the mass-to-charge ratio is determined using a quadrupole analyzer. For example, in a “quadrupole” or “quadrupole ion trap” instrument, ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between electrodes, the amplitude of the RF signal, and the mass/charge ratio. The voltage and amplitude may be selected so that only ions having a particular mass/charge ratio travel the length of the quadrupole, while all other ions are deflected. Thus, quadrupole instruments may act as both a “mass filter” and as a “mass detector” for the ions injected into the instrument.

One may enhance the resolution of the MS technique by employing “tandem mass spectrometry,” or “MS/MS”. In this technique, a precursor ion (also called a parent ion) generated from a molecule of interest can be filtered in an MS instrument, and the precursor ion subsequently fragmented to yield one or more fragment ions (also called daughter ions or product ions) that are then analyzed in a second MS procedure. By careful selection of precursor ions, only ions produced by certain analytes are passed to the fragmentation chamber, where collisions with atoms of an inert gas produce the fragment ions. Because both the precursor and fragment ions are produced in a reproducible fashion under a given set of ionization/fragmentation conditions, the MS/MS technique may provide an extremely powerful analytical tool. For example, the combination of filtration/fragmentation may be used to eliminate interfering substances, and may be particularly useful in complex samples, such as biological samples.

Alternate modes of operating a tandem mass spectrometric instrument include product ion scanning and precursor ion scanning. For a description of these modes of operation, see, e.g., E. Michael Thurman, et al., Chromatographic-Mass Spectrometric Food Analysis for Trace Determination of Pesticide Residues, Chapter 8 (Amadeo R. Fernandez-Alba, ed., Elsevier 2005) (387).

The results of an analyte assay may be related to the amount of the analyte in the original sample by numerous methods known in the art. For example, given that sampling and analysis parameters are carefully controlled, the relative abundance of a given ion may be compared to a table that converts that relative abundance to an absolute amount of the original molecule. Alternatively, external standards may be run with the samples, and a standard curve constructed based on ions generated from those standards. Using such a standard curve, the relative abundance of a given ion may be converted into an absolute amount of the original molecule. In certain preferred embodiments, an internal standard is used to generate a standard curve for calculating the quantity of steroidal compounds. Methods of generating and using such standard curves are well known in the art and one of ordinary skill is capable of selecting an appropriate internal standard. For example, in some embodiments, one or more isotopically labeled vitamin D metabolites (e.g., 25OHD₂-[6, 19, 19]-2_(H) ₃ and 25OHD₃-[6, 19, 19]-2_(H) ₃ ) may be used as internal standards. Numerous other methods for relating the amount of an ion to the amount of the original molecule will be well known to those of ordinary skill in the art.

One or more steps of the methods may be performed using automated machines. In certain embodiments, one or more purification steps are performed on-line, and more preferably all of the purification and mass spectrometry steps may be performed in an on-line fashion.

In certain mass spectrometry techniques, such as MS/MS, precursor ions are isolated for further fragmentation though collision activated dissociation (CAD). In CAD, precursor ions gain energy through collisions with an inert gas, and subsequently fragment by a process referred to as “unimolecular decomposition.” Sufficient energy must be deposited in the precursor ion so that certain bonds within the ion can be broken due to increased vibrational energy.

Steroidal compounds in a sample may be detected and/or quantified using MS/MS as follows. The samples may first be purified by protein precipitation or a hybrid protein precipitation/liquid-liquid extraction. Then, one or more steroidal compounds in the purified sample are derivatized with a Cookson-type reagent, such as PTAD or an isotopic variant thereof. The purified samples may then subjected to liquid chromatography, preferably on an extraction column (such as a TFLC column) followed by an analytical column (such as a HPLC column); the flow of liquid solvent from a chromatographic column enters the nebulizer interface of an MS/MS analyzer; and the solvent/analyte mixture is converted to vapor in the heated charged tubing of the interface. The analyte(s) (e.g., derivatized steroidal compounds such as derivatized vitamin D metabolites), contained in the solvent, are ionized by applying a large voltage to the solvent/analyte mixture. As the analytes exit the charged tubing of the interface, the solvent/analyte mixture nebulizes and the solvent evaporates, leaving analyte ions. Alternatively, derivatized steroidal compounds in the purified samples may not be subject to liquid chromatography prior to ionization. Rather, the samples may be spotted in a 96-well plate and volatilized and ionized via LDTD.

The ions, e.g. precursor ions, pass through the orifice of a tandem mass spectrometric (MS/MS) instrument and enter the first quadrupole. In a tandem mass spectrometric instrument, quadrupoles 1 and 3 (Q1 and Q3) are mass filters, allowing selection of ions (i.e., selection of “precursor” and “fragment” ions in Q1 and Q3, respectively) based on their mass to charge ratio (m/z). Quadrupole 2 (Q2) is the collision cell, where ions are fragmented. The first quadrupole of the mass spectrometer (Q1) selects for molecules with the mass to charge (m/z) ratios of derivatized steroidal compounds of interest. Precursor ions with the correct mass/charge ratios are allowed to pass into the collision chamber (Q2), while unwanted ions with any other mass/charge ratio collide with the sides of the quadrupole and are eliminated. Precursor ions entering Q2 collide with neutral argon gas molecules and fragment. The fragment ions generated are passed into quadrupole 3 (Q3), where the fragment ions of derivatized steroidal compounds of interest are selected while other ions are eliminated.

The methods may involve MS/MS performed in either positive or negative ion mode; preferably positive ion mode. Using standard methods well known in the art, one of ordinary skill is capable of identifying one or more fragment ions of a particular precursor ion of derivatized steroidal compounds that may be used for selection in quadrupole 3 (Q3).

As ions collide with the detector they produce a pulse of electrons that are converted to a digital signal. The acquired data is relayed to a computer, which plots counts of the ions collected versus time. The resulting mass chromatograms are similar to chromatograms generated in traditional HPLC-MS methods. The areas under the peaks corresponding to particular ions, or the amplitude of such peaks, may be measured and correlated to the amount of the analyte of interest. In certain embodiments, the area under the curves, or amplitude of the peaks, for fragment ion(s) and/or precursor ions are measured to determine the amount of a particular steroidal compounds. As described above, the relative abundance of a given ion may be converted into an absolute amount of the original analyte using calibration standard curves based on peaks of one or more ions of an internal molecular standard.

Processing Patient Samples for Analysis of Multiplex Patient Samples

Following the procedures outlined above, multiple patient samples can be multiplex (i.e., mixed and assayed together) if each patient sample is processed differently. The phrase “processed differently” means that each patient sample to be included in the multiplex sample is processed in such a way that steroidal compounds in two or more patient samples that are originally indistinguishable by mass spectrometry become distinguishable after processing. This may be accomplished by processing each patient sample with a different agent that derivitizes steroidal compounds. The derivatizing agents selected for use must generate derivatized steroidal compounds that are distinguishable by mass spectrometry. The basis for distinguishing derivatized steroidal compounds by mass spectrometry will be a difference in the mass of ions from the derivatized steroidal compounds. The differences in mass may arise from the use of two or more different derivatizing agents, such as PTAD and DMEQTAD. Differences in mass may also arise from the use of two or more isotopic variants of the same derivatizing agent, such as PTAD and 13_(C) ₆ -PTAD. These two approaches are not mutually exclusive, and any combination of different derivatizing agents and isotopic variants of the same agent may be used to uniquely label steroidal compounds in each patient sample in the plurality of patient samples to be analyzed. Optionally, one sample from the plurality of patient samples may be processed without a derivatizing agent.

After processing a plurality of patient samples, a particular steroidal compound from one patient sample will have a different mass spectrometric profile than the same steroidal compound in other patient samples. When processed patient samples are mixed to form a multiplex sample which is then analyzed to determine the levels of processed steroidal compounds, the differences in mass spectrometric profiles of the detected processed steroidal compounds allow for each processed steroidal compound to be attributed to an originating patient sample. Thus, the amounts of a steroidal compound in two or more patient samples are determined by a single mass spectrometric analysis of a multiplex sample.

As indicated above, different Cookson-type reagents may be used as derivatizing agents for different patient samples; for example, one patient sample may be derivatized with PTAD, and a second patient sample derivatized with DMEQTAD. Using different Cookson-type reagents generally results in large mass differences between the derivatized analytes. For example, the difference in mass between a steroidal compound derivatized with PTAD and the same compound derivatized with DMEQTAD is about 200 mass units (the mass difference between PTAD and DMEQTAD).

Isotopic variants of the same Cookson-type reagent may also be used to create distinguishable derivatives in multiple patient samples. For example, one patient sample may be derivatized with PTAD, and a second patient sample may be derivatized with 13_(C) ₆ -PTAD. In this example, the difference in mass between PTAD and 13_(C) ₆ -PTAD is about 6 mass units.

The following Examples serve to illustrate the invention through processing multiple patient samples with isotopic variants of PTAD. These Examples are in no way intended to limit the scope of the methods. In particular, the following Examples demonstrate quantitation of vitamin D metabolites by mass spectrometry with the use of 25OHD₂-[6, 19, 19]-2_(H) ₃ or 25OHD₃-[6, 19, 19]-2_(H) ₃ as internal standards. Demonstration of the methods of the present invention as applied to vitamin D metabolites does not limit the applicability of the methods to only vitamin D and vitamin D related compounds. Similarly, the use of 25OHD₂-[6, 19, 19]-2_(H) ₃ or 25OHD₃-[6, 19, 19]-2_(H) ₃ as internal standards are not meant to be limiting in any way. Any appropriate chemical species, easily determined by one in the art, may be used as an internal standard for steroidal compound quantitation.

EXAMPLES Example 1: Hybrid Protein Precipitation/Liquid-Liquid Extraction and Cookson-type Derivatization

The following automated hybrid protein precipitation/liquid-liquid extraction technique was conducted on patient serum samples. Gel Barrier Serum (i.e., serum collected in Serum Separator Tubes) as well as EDTA plasma and Heparin Plasma have also been established as acceptable for this assay.

A Perkin-Elmer Janus robot and a TomTec Quadra Tower robot was used to automate the following procedure. For each sample, 50 μL of serum was added to a well of a 96 well plate. Then 25 μL of internal standard cocktail (containing isotopically labeled 25OHD₂-[6, 19, 19]-2_(H) ₃ and 25OHD₃-[6, 19, 19]-2_(H) ₃ ) was added to each well, and the plate vortexed. Then 75 μL of methanol was added, followed by additional vortexing. 300 μL of ethyl acetate and 75 μL of water was then added, followed by additional vortexing, centrifugation, and transfer of the resulting supernatant to a new 96-well plate.

The transferred liquid in the second 96-well plate from Example 1 was dried to completion under a flowing nitrogen gas manifold. Derivatization was accomplished by adding 100 μL of a 0.1 mg/mL solution of the Cookson-type derivatization agent PTAD in acetonitrile to each well. The derivatization reaction was allowed to proceed for approximately one hour, and was quenched by adding 100 μL of water to the reaction mixture.

Example 2: Extraction of Vitamin D Metabolites with Liquid Chromatography

Sample injection was performed with a Cohesive Technologies Aria TX-4 TFLC system using Aria OS V 1.5.1 or newer software.

The TFLC system automatically injected an aliquot of the above prepared samples into a Cohesive Technologies C8XL online extraction column (50 μm particle size, 005×50 mm, from Cohesive Technologies, Inc.) packed with large particles. The samples were loaded at a high flow rate to create turbulence inside the extraction column. This turbulence ensured optimized binding of derivatized vitamin D metabolites to the large particles in the column and the passage of excess derivatizing reagent and debris to waste.

Following loading, the sample was eluted off to the analytical column, a Thermo Hypersil Gold Aq analytical column (5 μm particle size, 50×2.1 mm), with a water/ethanol elution gradient. The HPLC gradient was applied to the analytical column, to separate vitamin D metabolites from other analytes contained in the sample. Mobile phase A was water and mobile phase B was ethanol. The HPLC gradient started with a 35% organic gradient which was ramped to 99% in approximately 65 seconds.

Example 3: Detection and Quantitation of Derivatized Vitamin D Metabolites by MS/MS

MS/MS was performed using a Finnigan TSQ Quantum Ultra MS/MS system (Thermo Electron Corporation). The following software programs, all from Thermo Electron, were used in the Examples described herein: Quantum Tune Master V 1.5 or newer, Xcalibur V 2.07 or newer, LCQuan V 2.56 (Thermo Finnigan) or newer, and ARIA OS v1.5.1 (Cohesive Technologies) or newer. Liquid solvent/analyte exiting the analytical column flowed to the nebulizer interface of the MS/MS analyzer. The solvent/analyte mixture was converted to vapor in the tubing of the interface. Analytes in the nebulized solvent were ionized by ESI.

Ions passed to the first quadrupole (Q1), which selected ions for a derivatized vitamin D metabolite. Ions with a m/z of 570.32±0.50 were selected for PTAD-25OHD₂; ions with a m/z of 558.32±0.50 were selected for PTAD-25HD₃. Ions entering quadrupole 2 (Q2) collided with argon gas to generate ion fragments, which were passed to quadrupole 3 (Q3) for further selection. Mass spectrometer settings are shown in Table 1. Simultaneously, the same process using isotope dilution mass spectrometry was carried out with internal standards, PTAD-25OHD₂-[6, 19, 19]-2_(H) ₃ and PTAD-25OHD₃-[6, 19, 19]-2_(H) ₃ . The following mass transitions were used for detection and quantitation during validation on positive polarity. The indicated mass transitions re not meant to be limiting in any way. As seen in the Examples that follow, other mass transitions could be selected for each analyte to generate quantitative data.

TABLE 1 Mass Spectrometer Settings for Detection of PTAD-25OHD₂ and PTAD-25OHD₃. Mass Spectrometric Instrument Settings Discharge Current 4.0 μA Vaporizer Temperature 300 C. Sheath Gas Pressure 15 Ion Sweep Gas Pressure 0.0 Aux Gas Pressure 5 Capillary Temperature 300 C. Skimmer Offset −10 V Collision Pressure 1.5 mTorr Collision Cell Energy 15 V

TABLE 2 Exemplary Mass Transitions for PTAD-25OHD₂, PTAD-25OHD₂-[6,19,19]-²H₃ (IS), PTAD-25OHD₃, and PTAD-25OHD₃-[6,19,19]-²H₃ (IS) (Positive Polarity) Precursor Ion Production Analyte (m/z) (m/z) PTAD-25OHD₂ 570.32 298.09 PTAD-25OHD₂-[6,19,19]-²H₃ (IS) 573.32 301.09 PTAD-25OHD₃ 558.32 298.09 PTAD-25OHD₃-[6,19,19]-²H₃ (IS) 561.32 301.09

Exemplary chromatograms for PTAD-25OHD₃, PTAD-25OHD₃-[6, 19, 19]-2_(H) ₃ (IS), PTAD-25OHD₂, and PTAD-25OHD₂-[6, 19, 19]-2_(H) ₃ (IS) are found in FIGS. 1A, 1B, 1C, and 1D, respectively.

Exemplary calibration curves for the determination of 25OHD₂ and 25OHD₃ in serum specimens are shown in FIGS. 2A and 2B, respectively.

Example 4: Analytical Sensitivity: Lower Limit of Quantitation (LLOQ) and Limit of Detection (LOD)

The LLOQ is the point where measurements become quantitatively meaningful. The analyte response at this LLOQ is identifiable, discrete and reproducible with a precision (i.e., coefficient of variation (CV)) of greater than 20% and an accuracy of 80% to 120%. The LLOQ was determined by assaying five different human serum samples spiked with PTAD-25OHD₂ and PTAD-25OHD₃ at levels near the expected LLOQ and evaluating the reproducibility. Analysis of the collected data indicates that samples with concentrations of about 4 ng/mL yielded CVs of about 20%. Thus, the LLOQ of this assay for both PTAD-25OHD₂ and PTAD-25OHD₃ was determined to be about 4 ng/mL. The graphical representations of CV versus concentration for both analytes are shown in FIGS. 3A-B (FIG. 3A shows the plots over an expanded concentration range, while FIG. 3B shows the same plot expanded near the LOQ).

The LOD is the point at which a value is beyond the uncertainty associated with its measurement and is defined as three standard deviations from the zero concentration. To determine the LOD, generally, blank samples of the appropriate matrix are obtained and tested for interferences. However, no appropriate biological matrix could be obtained where the endogenous concentration of 25OHD₃ is zero, so a solution of 5% bovine serum albumin in phosphate buffered saline (with an estimated 1.5 ng/mL 25OHD₃) was used for LOD studies. The standard was run in 20 replicates each and the resulting area rations were statistically analyzed to determine that the LOD for 25OHD₂ and 25OHD₃ are about 1.9 and 3.3 ng/mL, respectively. Raw data from these studies is presented in Table 3, below

TABLE 3 Limit of Detection Raw Data and Analysis Replicate 25OHD₂ (ng/mL) 25OHD₃ (ng/mL) 1 0.0 0.0 2 1.1 2.0 3 0.1 2.4 4 0.3 1.1 5 0.5 1.9 6 0.4 1.8 7 0.2 1.9 8 0.5 2.3 9 1.1 2.3 10 0.5 2.1 11 0.4 1.5 12 1.2 1.9 13 0.4 1.8 14 0.3 1.6 15 0.0 1.3 16 0.9 1.3 17 0.8 1.5 18 0.1 1.9 19 0.5 1.7 20 0.4 1.8 Mean 0.4 1.7 SD 0.5 0.5 LOD (Mean + 3SD) 1.9 3.3

Example 5: Reportable Range and Linearity

Linearity of derivatized vitamin D metabolite detection in the assay was determined by diluting four pools serum with high endogenous concentration of either 25OHD₂ or 25OHD₃ and analyzing undiluted specimens and diluted specimens at 1:2, 1:4, and 1:8, in quadruplicate. Quadratic regression of the data was performed yielding correlation coefficients across the concentration range tested of R²=0.97. These studies demonstrated that specimens may be diluted at 1:4 with average recovery of 101%, permitting a reportable range of about 4 to about 512 ng/mL. Average measured values for each of the specimen dilution levels and correlation values from linear regression analysis are presented in Table 4A, below. Percent recoveries for each of the specimen dilution levels are presented in Table 4B, below.

TABLE 4A Linearity Data and Linear Regression Analysis over Reportable Range Dilution Level 25OHD₂ (ng/mL) 25OHD₃ (ng/mL) Pool 1 Pool 2 Pool 1 Pool 2 Undiluted 110.0 75.6 73.3 60.6 1:2 55.5 39.3 35.7 28.7 1:4 26.2 19.4 18.1 16.3 1:8 14.3 10.9 9.7 8.3 R² 0.9744 0.9721 0.9705 0.9601

TABLE 4B Percent Recovery at Various Specimen Dilution Levels Dilution Level 25OHD₂ (ng/mL) 25OHD₃ (ng/mL) Pool 1 Pool 2 Pool 1 Pool 2 Undiluted (100%) (100%) (100%) (100%) 1:2 100.9 104 97.4 94.8 1:4 95.5 102.7 98.6 107.3 1:8 104.2 115.0 106.0 109.0

Example 6: Analyte Specificity

The specificity of the assay against similar analytes was determined to have no cross reactivity for any vitamin D metabolite tested with the exception of 3-epi-25OHD₃, which behaves similarly to 25OHD₃ in the assay. The side-chain labeled stable isotopes of 25OHD₂ and 25OHD₃ also showed cross-reactivity owing to hydrogen exchange that occurs in the ion source. Thus, side-chain labeled stable isotopes of 25OHD₂ and 25OHD₃ should not be used as internal standards. Table 5, below, shows the compounds tested and the results of the cross-reactivity studies.

TABLE 5 Cross-Reactivity Studies (Compounds tested and results) Cross- Analyte 25OHD₂ 25OHD₃ Reactivity 1,25(OH)₂D₃ — — No 1,25(OH)₂D₂ — — No 1,25(OH)₂D₃-[6,19,19′]-²H — — No 1,25(OH)₂D₃-[26,26,26,27,27,27]-²H — — No 1,25(OH)₂D₂-[26,26,26,27,27,27]-²H — — No 25OHD₃ —  (100%) — 25OHD₂ (100%) — — 25OHD₃-IS-[6,19,19′]-²H — — No 25OHD₂-IS-[6,19,19′]-²H — — No 25OHD₃-IS-[26,26,26,27,27,27]-²H — 13.8% Yes 25OHD₂-IS-[26,26,26,27,27,27]-²H  2.7% — Yes vitamin D₃ — — No vitamin D₂ — — No vitamin D₃-[6,19,19′]-²H — — No vitamin D₂-[6,19,19′]-²H — — No vitamin D₃-[26,26,26,27,27,27]-²H — — No vitamin D₂-[26,26,26,27,27,27]-²H — — No 1-OH-D₃ (Alfacalcidiol) — — No 1-OH-D₂ (Hectoral) — — No 24,25(OH)₂D₃ — — No 25,26(OH)₂D₃ — — No 3-epi-25OHD₃ — — No 3-epi-1,25(OH)₂D₃ — 33.3% Yes Dihydrotachysterol — — No 1,25(OH)₂D₃-26,23-lactone — — No Paracalcitol (Zemplar) — — No Calcipotriene (Dovonex) — — No 7-Dehydrocholesterol — — No

Example 7: Reproducibility

Six standards at 5, 15, 30, 60, 90, and 120 ng/mL for each analyte were run in every assay as a means as quantitating reproducibility. The day-to-day reproducibility was determined using calibration curves from 19 assays. The data from these 19 assays are presented in Tables 6A (for 25OHD₂) and 6B (for 25OHD₃).

TABLE 6A Standard curves demonstrate reproducibility of 25OHD₂-PTAD determination. Concentration 5 15 30 60 90 120 Assay ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL 1 0.06 0.16 0.36 0.68 0.92 1.23 2 0.08 0.17 0.36 0.61 0.94 1.18 3 0.07 0.17 0.32 0.66 0.92 1.19 4 0.06 0.19 0.29 0.69 0.98 1.16 5 0.07 0.15 0.37 0.60 0.85 1.13 6 0.07 0.16 0.32 0.64 0.95 1.20 7 0.07 0.16 0.35 0.63 0.99 1.18 8 0.06 0.16 0.35 0.60 0.98 1.31 9 0.06 0.18 0.32 0.66 0.96 1.10 10 0.06 0.15 0.35 0.62 0.89 1.22 11 0.05 0.17 0.33 0.65 0.96 1.17 12 0.04 0.17 0.32 0.61 0.97 1.12 13 0.05 0.16 0.34 0.62 0.97 1.30 14 0.06 0.17 0.31 0.61 0.95 1.21 15 0.07 0.16 0.34 0.70 0.94 1.30 16 0.08 0.17 0.39 0.70 1.06 1.27 17 0.06 0.15 0.36 0.65 1.03 1.20 18 0.05 0.18 0.34 0.81 0.91 1.33 19 0.06 0.17 0.30 0.62 1.06 1.21 Avg 0.06 0.16 0.34 0.65 0.96 1.21 SD 0.01 0.01 0.02 0.05 0.05 0.07 CV % 15.4 6.3 7.4 8.0 5.6 5.4

TABLE 6B Standard curves demonstrate reproducibility of 25OHD₃-PTAD determination, Concentration 5 15 30 60 90 120 Assay ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL 1 0.07 0.16 0.36 0.61 0.95 1.19 2 0.07 0.17 0.32 0.66 1.01 1.12 3 0.06 0.16 0.32 0.60 1.00 1.16 4 0.06 0.17 0.31 0.60 0.94 1.09 5 0.05 0.16 0.33 0.65 0.96 1.11 6 0.07 0.17 0.34 0.65 0.87 1.13 7 0.07 0.17 0.31 0.61 0.95 1.21 8 0.06 0.15 0.29 0.58 0.90 1.21 9 0.07 0.17 0.32 0.65 0.88 1.15 10 0.06 0.14 0.30 0.57 1.05 1.16 11 0.06 0.15 0.30 0.56 0.87 1.15 12 0.05 0.15 0.31 0.64 0.85 1.06 13 0.06 0.16 0.33 0.60 0.88 1.08 14 0.06 0.17 0.31 0.61 0.91 1.22 15 0.06 0.18 0.34 0.66 0.96 1.18 16 0.06 0.17 0.35 0.65 0.94 1.21 17 0.06 0.17 0.36 0.64 0.94 1.17 18 0.07 0.17 0.34 0.66 0.98 1.18 19 0.07 0.16 0.34 0.68 0.84 1.27 Avg 0.06 0.16 0.33 0.63 0.93 1.16 SD 0.00 0.01 0.02 0.03 0.06 0.05 CV % 7.9 5.8 5.9 5.5 6.1 4.6

Example 8: Intra-assay and Inter-assay Variation Studies

Intra-assay variation is defined as the reproducibility of results for a sample within a single assay. To assess intra-assay variation, twenty replicates from each of four quality control (QC) pools covering the reportable range of the assay were prepared and measured from pooled serum with 25OHD₂ and 25OHD₃ at arbitrary ultralow, low, medium, and high concentrations for each analyte. Acceptable levels for the coefficient of variation (CV) are less then 15% for the three higher concentration, and less than 20% for the lowest concentration (at or near the LOQ for the assay).

The results of the intra-assay variation studies indicate that the CV for the four QC pools are 9.1%, 6.4%, 5.0%, and 5.9% with mean concentrations of 13.7 ng/mL, 30.0 ng/mL, 52.4 ng/mL, and 106.9 ng/mL, respectively, for PTAD-25OHD₂, and 3.5%, 4.9%, 5.1%, and 3.3% with mean concentrations of 32.8 ng/mL, 15.0 ng/mL, 75.4 ng/mL, and 102.3 ng/mL, respectively, for PTAD-25OHD₃. The data from analysis of these replicates is shown in Tables 7A and 7B.

TABLE 7A PTAD-25OHD₂ Intra-assay variation studies. QC (U) QC (L) QC (M) QC (H) Lot # Lot # Lot # Lot # 090837 090838 090839 090840 Replicate ng/mL ng/mL ng/mL ng/mL 1 15.2 31.4 49.5 108.9 2 12.3 29.7 53.2 109.3 3 13.8 30.8 50.9 98.9 4 12.4 30.1 50.4 111.5 5 14.6 27.2 49.7 109.0 6 14.6 29.1 47.6 110.3 7 13.6 33.0 53.3 95.6 8 11.4 29.9 53.3 98.5 9 14.0 31.5 55.2 110.7 10 13.7 29.1 49.0 113.5 11 13.7 29.5 56.8 100.4 12 13.0 25.5 54.1 105.4 13 15.6 34.2 53.6 102.0 14 11.7 28.7 52.9 103.2 15 13.5 28.1 49.4 121.0 16 13.6 29.8 52.0 102.9 17 13.1 29.4 56.8 113.4 18 14.4 30.6 54.5 103.3 19 16.2 31.6 53.1 110.8 20 12.7 30.7 — 110.4 Avg 0.06 0.16 0.33 0.63 SD 0.00 0.01 0.02 0.03 CV % 7.9 5.8 5.9 5.5

TABLE 7B PTAD-25OHD₃ Intra-assay variation studies. QC (U) QC (L) QC (M) QC (H) Lot # Lot # Lot # Lot # 090837 090838 090839 090840 Replicate ng/mL ng/mL ng/mL ng/mL 1 34.4 13.7 75.7 101.7 2 35.0 14.2 78.7 101.8 3 33.2 14.7 73.1 103.2 4 34.4 14.9 83.7 104.1 5 32.4 14.5 72.7 107.0 6 33.3 14.3 73.6 107.6 7 33.8 15.0 79.1 97.5 8 32.1 15.8 73.1 98.7 9 32.4 15.5 74.2 106.5 10 31.4 15.4 74.5 106.1 11 31.8 14.7 69.3 105.9 12 31.2 16.8 73.5 97.7 13 34.1 15.4 72.7 104.9 14 33.8 15.3 75.1 99.8 15 32.0 15.7 76.2 102.2 16 33.2 14.7 74.2 102.2 17 32.6 14.7 85.0 100.5 18 31.6 13.9 75.5 101.8 19 31.3 15.6 73.6 99.9 20 32.5 15.3 — 96.3 Avg 32.8 15.0 75.4 102.3 SD 1.1 0.7 3.8 3.4 CV % 3.5 4.9 5.1 3.3

Five aliquots of each of the same four QC pools were assayed over six days to determine the coefficient of variation (CV) between assays. The results of the intra-assay variation studies indicate that the inter-assay CV for the four QC pools are about 8.3%, 6.2%, 8.1%, and 6.4% with mean concentrations of about 13.1 ng/mL, 29.8 ng/mL, 51.9 ng/mL, and 107.8 ng/mL, respectively, for PTAD-25OHD₂, and about 4.8%, 6.7%, 4.7%, and 6.7% with mean concentrations of about 31.1 ng/mL, 14.5 ng/mL, 75.1 ng/mL, and 108.4 ng/mL, respectively, for PTAD-25OHD₃. The data from analysis of these replicates is shown in Tables 8A and 8B.

TABLE 8A PTAD-25OHD₂ Inter-assay variation studies. QC (U) QC (L) QC (M) QC (H) Lot # Lot # Lot # Lot # 090837 090838 090839 090840 Assay ng/mL ng/mL ng/mL ng/mL 1 13.6 28.1 51.7 119.6 12.8 30.1 49.4 117.9 14.6 32.0 49.7 105.1 13.0 30.8 52.3 100.2 13.0 29.2 56.6 110.3 2 12.9 31.3 46.3 108.1 13.5 30.3 52.1 117.8 10.9 29.7 46.9 105.8 11.2 30.6 43.6 105.2 12.8 28.7 50.3 104.9 3 12.6 28.8 56.5 115.3 16.4 29.3 63.8 103.0 13.2 26.2 45.5 103.2 11.5 30.8 53.8 113.2 12.4 33.7 51.6 106.9 4 12.1 28.5 58.5 97.0 13.9 26.2 51.8 115.1 14.4 29.6 48.9 112.2 13.1 32.1 52.3 97.9 12.6 30.5 52.2 104.2 5 12.7 29.9 54.5 101.3 14.3 28.3 46.3 102.2 13.9 30.0 56.1 111.4 13.1 32.6 51.2 123.1 12.4 26.2 51.2 98.3 6 12.5 30.6 50.1 104.6 12.9 32.6 51.8 104.8 14.0 28.6 53.7 108.9 14.3 29.1 51.0 113.8 12.9 29.1 56.4 102.2 Avg 13.1 29.8 51.9 107.8 SD 1.1 1.8 4.2 6.8 CV % 8.3 6.2 8.1 6.4

TABLE 8B PTAD-25OHD₃ Inter-assay variation studies. QC (U) QC (L) QC (M) QC (H) Lot # Lot # Lot # Lot # 090837 090838 090839 090840 Assay ng/mL ng/mL ng/mL ng/mL 1 32.6 13.4 76.7 104.9 30.0 12.7 77.6 107.0 34.1 15.4 78.4 107.1 34.0 14.8 76.6 105.1 30.2 15.5 74.8 110.2 2 33.5 13.2 69.8 109.8 32.4 14.3 75.0 106.4 30.2 16.2 73.4 112.1 31.4 16.1 71.9 97.0 31.4 13.7 75.2 117.5 3 31.5 13.3 70.2 112.4 32.1 14.6 82.6 101.5 31.0 15.4 70.8 99.8 28.7 15.6 74.3 103.6 30.7 15.1 79.8 99.1 4 31.9 14.5 76.3 124.2 27.5 14.0 70.5 113.6 27.9 14.8 74.5 112.5 32.1 16.1 74.3 108.8 31.0 14.4 74.5 110.1 5 31.2 13.1 76.7 96.5 31.5 13.5 82.9 106.1 31.5 14.7 70.9 112.9 30.9 14.5 77.6 117.7 31.0 13.9 73.1 101.9 6 29.8 15.6 73.3 110.1 30.5 13.5 71.5 99.3 31.0 13.9 72.6 120.5 30.5 14.6 74.2 109.4 30.7 13.6 81.8 115.9 Avg 31.1 14.5 75.1 108.4 SD 1.5 1.0 3.6 6.9 CV % 4.8 6.7 4.7 6.4

Example 9: Recovery Studies

Two recovery studies were performed. The first was performed using six specimens, spiked with two different concentrations each of 25OHD₂ and 25OHD₃. These spiked specimens were subjected to the hybrid protein precipitation/liquid-liquid extraction procedure described in Example 1. Then, aliquots of the extracts of the spiked specimens were derivatized with normal PTAD, following the procedure discussed above, and analyzed in quadruplicate. The spiked concentrations were within the workable range of the assay. The six pools yielded an average accuracy of about 89% at spiked levels of greater than about 44 ng/mL and about 92% at spiked levels of greater than about 73 ng/mL. Only two of the 24 experimental recoveries were less than 85%; the remaining 22 assays were within the acceptable accuracy range of 85-115%. The results of the spiked specimen recovery studies are presented in Table 9, below.

TABLE 9 Spiked Specimen Recovery Studies 25OHD₂ 25OHD₃ (% (% ng/ Recov- ng/ Recov- Pool Spike Level mL ery) mL ery) 1 — 12.0 — 10.8 — 44 ng/mL 25OHD₂ 48.0 81.2 10.7 — 73 ng/mL 25OHD₂ 79.0 91.6 10.7 — 44 ng/mL 25OHD₃ 12.7 — 51.9 92.9 73 ng/mL 25OHD₃ 11.5 — 76.5 89.9 2 — 11.9 — 10.8 — 44 ng/mL 25OHD₂ 48.0 81.4 10.6 — 73 ng/mL 25OHD₂ 75.6 87.1 11.0 — 44 ng/mL 25OHD₃ 10.0 — 48.8 85.6 73 ng/mL 25OHD₃ 11.6 — 76.4 89.7 3 — 13.6 — 6 — 44 ng/mL 25OHD₂ 52.5 87.8 10.9 — 73 ng/mL 25OHD₂ 76.8 86.4 10.5 — 44 ng/mL 25OHD₃ 13.2 — 49.6 88.0 73 ng/mL 25OHD₃ 12.3 — 78.0 92.2 4 — 9.0 — 12.7 — 44 ng/mL 25OHD₂ 50.3 93.1 13.5 — 73 ng/mL 25OHD₂ 77.6 93.8 13.2 — 44 ng/mL 25OHD₃ 10.0 — 52.1 89.0 73 ng/mL 25OHD₃ 9.5 — 83.6 97.0 5 — 21.8 — 14.0 — 44 ng/mL 25OHD₂ 68.0 104.2  13.3 — 73 ng/mL 25OHD₂ 91.1 94.8 13.6 — 44 ng/mL 25OHD₃ 23.3 — 53.5 89.1 73 ng/mL 25OHD₃ 22.2 — 86.4 99.1 6 — 13.8 — 9.3 — 44 ng/mL 25OHD₂ 50.6 83.0 9.2 — 73 ng/mL 25OHD₂ 83.9 95.9 9.5 — 44 ng/mL 25OHD₃ 13.5 — 48.6 88.6 73 ng/mL 25OHD₃ 13.2 — 76.5 91.9

The second recovery study was performed again using six specimens. Of these six specimens, three had high endogenous concentration of 25OHD₂ and three had high endogenous concentrations of 25OHD₃. The specimens were paired and mixed at ratios of about 4:1, 1:1, and 1:4. The resulting mixtures were subjected to the hybrid protein precipitation/liquid-liquid extraction procedure described in Example 1. Then, aliquots of the extracts of the mixed specimens were derivatized with normal PTAD, following the procedure discussed above, and analyzed in quadruplicate. These experiments yielded an average accuracy of about 98% for 25OHD₂ and about 93% for 25OHD₃. All individual results were within the acceptable accuracy range of 85-115%. The results of the mixed specimen recovery studies are presented in Table 10, below.

TABLE 10 Mixed Specimen Recovery Studies 25OHD₂ 25OHD₃ Mea- Ex- Recov- Mea- Ex- Recov- Specimen sured pected ery sured pected ery Mixture ng/mL ng/mL (%) ng/mL ng/mL (%) 100% A 45.2 — — 5.5 — — 4:1 A:B 37.1 37.0 100  11.6 13.1 88 1:1 A:B 26.4 24.6 107  24.4 24.4 100  1:4 A:B 12.6 12.3 102  33.9 35.7 95 100% B 4.1 — — 43.3 — — 100% C 46.8 — — 8.3 — — 4:1 C:D 38.1 38.7 98 17.7 18.3 97 1:1 C:D 25.0 26.6 94 32.0 33.4 96 1:4 C:D 14.4 14.4 100  46.5 48.4 96 100% D 6.3 — — 58.5 — — 100% E 38.7 — — 7.4 — — 4:1 E:F 33.4 34.3 97 15.7 17.5 89 1:1 E:F 27.1 27.7 98 27.8 32.6 85 1:4 E:F 18.3 21.0 87 44.0 47.7 92 100% F 16.6 — — 57.8 — — *Measured values are averages of analysis of four aliquots.

Example 10: Method Correlation Study

The method of detecting vitamin D metabolites following PTAD-derivatization was compared to a mass spectrometric method in which the vitamin D metabolites are not derivatized prior to analysis. Such a method is described in the published U.S. Patent Application 2006/0228808 (Caulfield, et al.). Eight specimens were split and analyzed according to both methods. The correlation between the two methods was assessed with linear regression, deming regression, and Bland-Altman analysis for complete data sets (including calibration samples, QC pools, and unknowns), as well as for unknowns only.

Plots of the linear regression analysis and the Deming regression analysis are shown in FIGS. 4A-B (for 25OHD₂) and FIGS. 5A-B (for 25OHD₃).

Example 11: Hemolysis, Lipemia, and Icteria Studies

The effect hemolysis, lipemia, and icteria have on the assay was also investigated.

Hemolysis. The effect of hemolysis was evaluated by pooling patient samples with known endogenous concentrations of both 25OHD₂ and 25OHD₃ to create five different pools with concentrations across the dynamic range of the assay. Then, lysed whole blood was spiked into the pools to generate lightly and moderately hemolyzed samples.

The lightly and moderately hemolyzed samples were analyzed in quadruplicate and the results were compared to levels of samples without whole blood spikes. The resulting comparison indicated a % difference of less than 15% for both 25OHD₂ and 25OHD₃. Therefore, light to moderately hemolyzed specimens are acceptable for analysis.

Lipemia. The effect of lipemia was evaluated by pooling patient samples with known endogenous concentrations of both 25OHD₂ and 25OHD₃ to create five different pools with concentrations across the dynamic range of the assay. Then, powdered lipid extract was added to the pools to generate lightly and grossly lipemic specimens. Specimens were run in quadruplicate and results were compared to the non-lipemic pool result and the accuracy was calculated. The data shows that determination of 25OHD₂ is unaffected by lipemia (all values were within an acceptable accuracy range of 85-115%), however, 25OHD₃ is affected by lipemia, resulting in determination in lower than expected values. The degree of variance increased with the degree of lipemia. Therefore, light but not grossly lipemic specimens are acceptable.

Icteria. The effect of icteria was evaluated by pooling patient samples with known endogenous concentrations of both 25OHD₂ and 25OHD₃ to create five different pools with concentrations across the dynamic range of the assay. Then, a concentrated solution of Bilirubin was spiked into the pools to generate lightly and grossly icteric specimens. Specimens were run in quadruplicate and results were compared to the non-icteric pool result and the accuracy was calculated. The data showed that 25OHD₂ and 25OHD₃ are unaffected by icteria (with all values within an acceptable accuracy range of 85-115%). Therefore, icteric specimens are acceptable.

Example 12: Injector Carryover Studies

Blank matrices were run immediately after a specimen with a high concentration of 25OHD₂ and 25OHD₃ in order to evaluate carryover between samples. These studies indicated that the response at the retention time of analyte or internal standard was not large enough to compromise the integrity of the assay. Data from these studies is presented in Table 11, below.

TABLE 11 Injector Carryover Study Results Specimen 25OHD₂ 25OHD₃ Injection Type (ng/mL) (ng/mL) 1 Blank 0.9 1.6 2 High 292.6 356.8 3 Blank 1.0 0.9 4 Blank −0.1 0.5 5 High 290.1 360.1 6 High 299.9 350.5 7 Blank 1.0 1.5 8 Blank 0.6 1.4 9 Blank 1.3 1.4 10 High 285.8 352.1 11 High 303.1 312.1 12 High 293.8 295.1 13 Blank 0.9 0.8 14 Blank 1.0 1.8 15 Blank 1.1 1.4 16 Blank 1.0 1.6 17 High 291.7 371.6 18 High 334.2 360.1 19 High 301.7 328.5 20 High 283.1 382.1 21 Blank 0.6 1.1 22 Blank 0.6 1.3 23 Blank 0.7 1.4 24 Blank 0.4 1.9 25 Blank 0.4 0.9 26 High 300.7 311.7 27 High 279.5 302.0 28 High 317.5 341.0 29 High 261.5 403.4 30 High 288.3 362.6 31 Blank 2.7 1.6 32 Blank 1.7 1.2 33 Blank 0.5 1.3 34 Blank 1.3 1.7 35 Blank 0.3 1.6 36 Blank 0.6 1.4 37 High 311.7 366.2 38 High 314.1 342.0 39 High 325.7 349.1 40 High 289.6 326.6 41 High 291.5 322.3 42 High 278.9 336.5 43 Blank 2.1 2.5 44 Blank 0.6 1.6 45 Blank 0.7 1.4 46 Blank 0.7 1.5 47 Blank 0.1 1.0 48 Blank 0.7 1.1 49 Blank 1.3 1.0 50 High 281.2 345.6 51 High 312.5 348.3 52 High 304.8 329.1 53 High 290.5 353.9 54 High 286.4 344.9 55 High 302.5 330.6 56 High 292.2 388.5 57 Blank 0.8 1.5 58 Blank 1.3 1.4 59 Blank 3.5 2.6 60 Blank 0.4 1.8 61 Blank 1.0 1.4 62 Blank 1.0 1.2 63 Blank 0.7 1.0 64 Blank 1.1 1.4 65 High 285.4 355.4 66 High 318.0 355.0 67 High 285.5 345.7 68 High 303.0 317.1 69 High 276.3 351.4 70 High 321.8 350.4 71 High 279.4 329.6 72 High 299.1 337.9 73 Blank 0.9 1.6 74 Blank 1.7 1.6 75 Blank 1.0 1.1 76 Blank 1.8 2.7 77 Blank 1.0 1.9 78 Blank 0.6 1.1 79 Blank 0.9 0.9 80 Blank 1.2 2.2

Example 13: Suitable Specimen Types

The assay was conducted on various specimen types. Human serum and Gel-Barrier Serum (i.e., serum from Serum Separator Tubes), as well as EDTA Plasma and Heparin were established as acceptable sample types. In these studies, sets of human serum (serum), Gel-Barrier Serum (SST), EDTA Plasma (EDTA), and heparin (Na Hep) drawn at the same time from the same patient were tested for 25OHD₂ (40 specimen sets) and 25OHD₃ (6 specimen sets). Due to the limitations with clot detection/sensing in existing automated pipetting systems, plasma was not tested for automated procedures.

The results of the specimen type studies are presented in Tables 12A and B for 25OHD₂ and 25OHD₃, respectively.

TABLE 12A Results from Specimen Type Studies for 25OHD₂ Specimen CC Measured Concentration 25OHD₂ (ng/mL) Set ID# Serum SST EDTA Na Hep 1 5804 26.8 25.7 24.3 26.8 2 5207 16.1 17.6 16.1 16.5 3 5235 17.4 17.7 16.8 17.2 4 5333 62.9 62.7 63.7 57.4 5 5336 33.0 32.4 28.8 28.8 6 5339 17.2 17.6 17.8 17.8 7 5340 16.7 17.1 16.8 16.5 8 5342 28.6 27.9 26.9 30.5 9 5344 23.3 23.8 22.3 22.9 10 5351 19.4 20.0 20.4 21.4 11 5355 17.6 16.7 19.4 18.3 12 5362 25.3 25.2 23.5 24.0 13 5365 40.9 44.7 46.8 42.9 14 5406 23.1 20.3 21.5 20.5 15 5408 31.7 33.9 31.6 32.3 16 5414 21.1 21.8 21.2 20.4 17 5422 44.0 47.7 45.5 47.3 18 5432 13.6 14.2 12.3 13.8 19 5463 15.1 15.4 15.6 14.5 20 5493 38.6 42.2 40.1 36.8 21 5366 47.5 48.1 46.7 45.1 22 5368 23.0 23.6 22.3 22.3 23 5392 34.1 33.4 34.4 27.6 24 5451 36.4 42.1 40.0 38.3 25 5455 27.3 29.9 25.1 27.9 26 5476 16.7 17.9 15.8 16.6 27 5483 30.4 28.2 26.5 28.1 28 5484 38.2 37.7 37.2 36.0 29 5537 30.5 30.3 27.2 27.0 30 5547 9.2 9.0 8.7 8.2 31 5560 9.4 10.9 9.8 8.6 32 5571 30.9 31.7 29.6 29.2 33 5572 47.6 50.3 47.7 48.6 34 5577 11.2 11.7 10.4 9.2 35 5606 39.3 38.8 41.0 37.7 36 5611 21.9 25.3 20.7 21.1 37 5650 38.0 34.3 34.6 36.2 38 5651 34.8 32.8 32.4 32.4 39 5653 29.4 32.3 28.1 27.0 40 5668 11.4 12.8 14.2 13.1

TABLE 12B Results from Specimen Type Studies for 25OHD₃ Specimen CC Measured Concentration 25OHD₃ (ng/mL) Set ID# Serum SST EDTA Na Hep 2 5207 6.6 6.9 7.1 7.2 6 5339 5.8 5.2 4.5 5.6 11 5355 7.8 8.2 8.8 8.2 20 5493 3.9 4.2 4.3 4.2 37 5650 3.7 4.5 4.6 5.2 39 5653 4.7 5.1 4.6 4.7

Example 14: Multiplex Patient Samples with Multiple Derivatizing Agents

Patient sample multiplexing after derivatization with different derivatizing agents was demonstrated in the following crossover experiments.

First, two patients samples (i.e., sample A and sample B) were both subjected to the hybrid protein precipitation/liquid-liquid extraction procedure described in Example 1. Then, aliquots of the extracts from sample A and sample B were derivatized with normal PTAD, following the procedure discussed above. Second aliquots of the extracts from sample A and sample B were also derivatized with 13_(C) ₆ -PTAD, also according to the procedure discussed above.

After the four derivatization reactions were quenched, a portion of the PTAD-derivatized sample A was mixed with 13_(C) ₆ -PTAD-derivatized sample B, and a portion of 13_(C) ₆ -PTAD-derivatized sample A was mixed with PTAD-derivatized sample B.

These mixtures were loaded onto a 96-well plate and analyzed according to the liquid chromatography-mass spectrometry methods described in Examples 2 and 3. Again, 25OHD₂-[6, 19, 19]-2_(H) ₃ and 25OHD₃-[6, 19, 19]-2_(H) ₃ were used as internal standards (shown in Table 13, below, as 25OHD₂-IS and 25OHD₃-IS). The mass spectrometer was programmed to monitor for the PTAD- and 13_(C) ₆ -PTAD-derivatized vitamin D metabolite conjugates shown in Table 13. The indicated mass transitions re not meant to be limiting in any way. As seen in the Examples that follow, other mass transitions could be selected for each analyte to generate quantitative data.

TABLE 13 Ions monitored for mass spectrometric determination of multiplex PTAD- and ¹³C₆-PTAD-derivatized samples (by MRM). Analyte Precursor Fragment PTAD-25OHD₃ 558 298 PTAD-25OHD₃-IS 561 301 PTAD-25OHD₂ 570 298 PTAD-25OHD₂-IS 573 301 ¹³C₆-PTAD-25OHD₃ 564 304 ¹³C₆-PTAD-25OHD₃-IS 567 307 ¹³C₆-PTAD-25OHD₂ 576 304 ¹³C₆-PTAD-25OHD₂-IS 579 307

Derivatized samples A and B and permutations of mixtures of the two described above were analyzed and plotted to evaluate goodness of fit of the data. These results are presented in FIGS. 6A-D, 7A-D, and 8A-D.

FIGS. 6A-D are plots comparing the results of analysis of multiplex samples and unmixed samples (with the same derivatization agent). These plots show R² values in excellent agreement (i.e., R² values for all four variants are in excess of 0.98). This shows that, given a constant derivatization agent, analysis of mixed samples gives the same result as analysis of unmixed samples.

FIGS. 7A-D are plots comparing the results of analysis of the same specimen treated with different derivatization agents (but comparing mixed versus mixed, or unmixed versus unmixed samples). These plots also show R² values in excellent agreement (i.e., R² values for all four variants are in excess of 0.98). This shows that the isotopic variation between PTAD and 13_(C) ₆ -PTAD is not a source of difference in the performance of the assay, at least when the compared samples are both mixed, or unmixed.

FIGS. 8A-D are plots comparing the results of analysis of the same specimen treated with different derivatization agents, with one analysis coming from a mixed sample and one coming from an unmixed sample. These plots also show R² values in excellent agreement (i.e., R² values for all four variants are in excess of 0.99). This shows that the isotopic variation between PTAD and 13_(C) ₆ -PTAD in combination with variation between mixed and unmixed samples is not a source of difference in the performance of the assay.

Thus, isotopic variation of the PTAD derivatization agent made no meaningful difference even when samples were mixed together and introduced into the mass spectrometer as a single injection. Multiplexing of patient samples was successfully demonstrated.

Example 15: Exemplary Spectra from LDTD-MS/MS Analysis of Native and PTAD Derivatized 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃

Underivatized and PTAD derivatized 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ were analyzed by LDTD-MS/MS. Results of these analyses are presented below.

Exemplary Q1 scan spectra from analysis of 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ are shown in FIGS. 9A and 10A, respectively. These spectra were collected by scanning Q1 across a m/z range of about 350 to 450.

Exemplary product ion scans from each of these species are presented in FIGS. 9B and 10B, respectively. The precursor ions selected in Q1, and collision energies used in fragmenting the precursors are indicated in Table 14.

A preferred MRM transition for the quantitation of 25-hydroxyvitamin D₂ is fragmenting a precursor ion with a m/z of about 395.2 to a product ion with a m/z of about 208.8 or 251.0. A preferred MRM transition for the quantitation of 25-hydroxyvitamin D₃ is fragmenting a precursor ion with a m/z of about 383.2 to a product ion with a m/z of about 186.9 or 257.0. However, as can be seen in the product ion scans in FIGS. 9B and 10B, additional product ions may be selected to replace or augment the preferred fragment ion.

TABLE 14 Precursor Ions and Collision Cell Energies for Fragmentation of 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ Precursor Ion Collision Cell Energy Analyte (m/z) (V) 25-hydroxyvitamin D₂ 395.2 20 25-hydroxyvitamin D₃ 383.2 20

Exemplary Q1 scan spectra from the analysis of samples containing PTAD-25-hydroxyvitamin D₂ and PTAD-25-hydroxyvitamin D₃ are shown in FIGS. 11A and 12A, respectively. These spectra were collected by scanning Q1 across a m/z range of about 520 to 620.

Exemplary product ion scans from each of these species are presented in FIGS. 11B and 12B, respectively. The precursor ions selected in Q1, and collision energies used in fragmenting the precursors are indicated in Table 15.

A preferred MRM transition for the quantitation of PTAD-25-hydroxyvitamin D₂ is fragmenting a precursor ion with a m/z of about 570.3 to a product ion with a m/z of about 298.1. A preferred MRM transition for the quantitation of PTAD-25-hydroxyvitamin D₃ is fragmenting a precursor ion with a m/z of about 558.3 to a product ion with a m/z of about 298.1. However, as can be seen in the product ion scans in FIGS. 11B and 12B, additional product ions may be selected to replace or augment the preferred fragment ion.

TABLE 15 Precursor Ions and Collision Cell Energies for Fragmentation of PTAD-25-hydroxyvitamin D₂ and PTAD-25-hydroxyvitamin D₃ Precursor Ion Collision Cell Energy Analyte (m/z) (V) PTAD-25- 570.3 15 hydroxyvitamin D₂ PTAD-25- 558.3 15 hydroxyvitamin D₃

Example 16: Exemplary Spectra from LDTD-MS/MS Analysis of PTAD Derivatized 1α,25-dihydroxyvitamin D₂ and 1α,25-dihydroxyvitamin D₃

PTAD derivatives of 1α,25-dihydroxyvitamin D₂ and 1α,25-dihydroxyvitamin D₃ were prepared by treating aliquots of stock solutions of each analyte with PTAD in acetonitrile. The derivatization reactions was allowed to proceed for approximately one hour, and were quenched by adding water to the reaction mixture. The derivatized analytes were then analyzed according to the LDTD-MS/MS procedure outlined above.

Exemplary Q1 scan spectra from the analysis of samples containing PTAD-1α,25-dihydroxyvitamin D₂ and PTAD-1α,25-hydroxyvitamin D₃ are shown in FIGS. 13A, and 14A, respectively. These spectra were collected with LDTD-MS/MS by scanning Q1 across a m/z range of about 520 to 620.

Exemplary product ion scans generated from three different precursor ions for each of PTAD-1α,25-dihydroxyvitamin D₂ and PTAD-1α,25-hydroxyvitamin D₃ are presented in FIGS. 13B-D, and 14B-D, respectively. The precursor ions selected in Q1 and the collision energies used to generate these product ion spectra are indicated in Table 16.

Exemplary MRM transitions for the quantitation of PTAD-1α,25-dihydroxyvitamin D₂ include fragmenting a precursor ion with a m/z of about 550.4 to a product ion with a m/z of about 277.9; fragmenting a precursor ion with a m/z of about 568.4 to a product ion with a m/z of about 298.0; and fragmenting a precursor ion with a m/z of about 586.4 to a product ion with a m/z of about 314.2. Exemplary MRM transitions for the quantitation of PTAD-1α,25-hydroxyvitamin D₃ include fragmenting a precursor ion with a m/z of about 538.4 to a product ion with a m/z of about 278.1; fragmenting a precursor ion with a m/z of about 556.4 to a product ion with a m/z of about 298.0; and fragmenting a precursor ion with a m/z of about 574.4 to a product ion with a m/z of about 313.0. However, as can be seen in the product ion scans in FIGS. 6B-D and 7B-D, several other product ions are generated upon fragmentation of the precursor ions. Additional product ions may be selected from those indicated in FIGS. 13B-D and 14B-D to replace or augment the exemplary fragment ions.

TABLE 16 Precursor Ions and Collision Cell Energies for Fragmentation of PTAD-1α,25-dihydroxyvitamin D₂ and PTAD-1α,25- dihydroxyvitamin D3 Precursor Ion Energy of Collision Cell Analyte (m/z) (V) PTAD-1α,25- 550.4, 568.4, 15 dihydroxyvitamin D₂ 586.4 PTAD-1α,25- 538.4, 556.4, 15 dihydroxyvitamin D₃ 574.4

PTAD derivatives of various deuterated forms of dihydroxyvitamin D metabolites were also investigated. PTAD derivatives of 1α,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-2_(H) ₆ , 1α,25-dihydroxyvitamin D₃-[6, 19, 19]-2_(H) ₃ , and 1α,25-dihydroxyvitamin D₃-[26, 26, 26, 27, 27, 27]-2_(H) ₆ were prepared and analyzed as above.

Exemplary MRM transitions for the quantitation of PTAD-1α,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-2_(H) ₆ include fragmenting a precursor ion with a m/z of about 556.4 to a product ion with a m/z of about 278.1; fragmenting a precursor ion with a m/z of about 574.4 to a product ion with a m/z of about 298.1; and fragmenting a precursor ion with a m/z of about 592.4 to a product ion with a m/z of about 313.9.

Exemplary MRM transitions for the quantitation of PTAD-1α,25-dihydroxyvitamin D₃-[6, 19, 19]-2_(H) ₃ include fragmenting a precursor ion with a m/z of about 541.4 to a product ion with a m/z of about 280.9; fragmenting a precursor ion with a m/z of about 559.4 to a product ion with a m/z of about 301.1; and fragmenting a precursor ion with a m/z of about 577.4 to a product ion with a m/z of about 317.3. Exemplary MRM transitions for the quantitation of PTAD-1α,25-dihydroxyvitamin D₂-[26, 26, 26, 27, 27, 27]-2_(H) ₆ include fragmenting a precursor ion with a m/z of about 544.4 to a product ion with a m/z of about 278.0; fragmenting a precursor ion with a m/z of about 562.4 to a product ion with a m/z of about 298.2; and fragmenting a precursor ion with a m/z of about 580.4 to a product ion with a m/z of about 314.0.

Example 17: Exemplary Spectra from MS/MS Analysis of PTAD Derivatized Vitamin D₂ and Vitamin D₃

PTAD derivatives of vitamin D₂, and vitamin D₃ were prepared by treating aliquots of stock solutions of each analyte with PTAD in acetonitrile. The derivatization reactions was allowed to proceed for approximately one hour, and were quenched by adding water to the reaction mixture. The derivatized analytes were then analyzed by MS/MS.

Exemplary Q1 scan spectra from the analysis of samples containing PTAD-vitamin D₂, and PTAD-vitamin D₃ are shown in FIGS. 15A and 16A, respectively. These analyses were conducted by directly injecting standard solutions containing the analyte of interest into a Finnigan TSQ Quantum Ultra MS/MS system (Thermo Electron Corporation). A liquid chromatography mobile phase was simulated by passing 800 μL/min of 80% acetonitrile, 20% water with 0.1% formic acid through an HPLC column, upstream of introduction of the analyte. The spectra were collected by scanning Q1 across a m/z range of about 500 to 620.

Exemplary product ion scans generated from precursor ions for each of PTAD-vitamin D₂ and PTAD-vitamin D₃ are presented in FIGS. 15B and 16B, respectively. The precursor ions selected in Q1 and the collision energies used to generate these product ion spectra are indicated in Table 17.

An exemplary MRM transition for the quantitation of PTAD-vitamin D₂ includes fragmenting a precursor ion with a m/z of about 572.2 to a product ion with a m/z of about 297.9. An exemplary MRM transition for the quantitation of PTAD-vitamin D₃ includes fragmenting a precursor ion with a m/z of about 560.2 to a product ion with a m/z of about 298.0. However, as can be seen in the product ion scans in FIGS. 15B and 16B, several other product ions are generated upon fragmentation of the precursor ions. Additional product ions may be selected from those indicated in FIGS. 15B and 16B to replace or augment the exemplary fragment ions.

TABLE 17 Precursor Ions and Collision Cell Energies for Fragmentation of PTAD-vitamin D₂ and PTAD-vitamin D₃ Precursor Ion Collision Cell Energy Analyte (m/z) (V) PTAD-vitamin D₂ 572.2 15 PTAD-vitamin D₃ 560.2 15

PTAD derivatives of various deuterated forms of vitamin D were also investigated. PTAD derivatives of vitamin D₂-[6, 19, 19]-2_(H) _(3,) vitamin D₂-[26, 26, 26, 27, 27, 27]-2_(H) ₆ , vitamin D₃-[6, 19, 19]-2_(H) ₃ , and vitamin D₃-[26, 26, 26, 27, 27, 27]-2_(H) ₆ were prepared and analyzed as above.

An exemplary MRM transition for the quantitation of PTAD-vitamin D₂-[6, 19, 19]-2_(H) ₃ includes fragmenting a precursor ion with a m/z of about 575.2 to a product ion with a m/z of about 301.0. An exemplary MRM transition for the quantitation of PTAD-vitamin D₂-[26, 26, 26, 27, 27, 27]-2_(H) ₆ includes fragmenting a precursor ion with a m/z of about 578.2 to a product ion with a m/z of about 297.9.

An exemplary MRM transition for the quantitation of PTAD-vitamin D₃-[6, 19, 19]-2_(H) ₃ includes fragmenting a precursor ion with a m/z of about 563.2 to a product ion with a m/z of about 301.0. An exemplary MRM transition for the quantitation of PTAD-vitamin D₃-[26, 26, 26, 27, 27, 27]-2_(H) ₆ includes fragmenting a precursor ion with a m/z of about 566.2 to a product ion with a m/z of about 298.0.

The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. It is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the invention embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the methods. This includes the generic description of the methods with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, where features or aspects of the methods are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. 

That which is claimed is:
 1. A method for determining the amount of one or more vitamin D metabolites in each of a plurality of human samples with a single tandem mass spectrometric assay, the method comprising: i) subjecting each of a plurality of human samples to a different isotopic variant of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) to generate a differently derivatized one or more vitamin D metabolites in each of the plurality of samples; ii) combining the plurality of samples to form a multiplex sample; and iii) quantifying the amount of the one or more vitamin D metabolites in each sample by mass spectrometry.
 2. The method of claim 1, further comprising subjecting additional samples to different Cookson-type derivatization agents selected from the group consisting of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), 4-methyl-1,2,4-triazoline-3,5-dione (MTAD), 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQTAD), 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD), 4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD), and isotopic variants thereof.
 3. The method of claim 1, wherein the plurality of samples comprises two samples, wherein a first derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) and a second derivatizing reagent is ¹³C₆-4-phenyl-1,2,4-triazoline-3,5-dione (¹³C₆-PTAD).
 4. The method of claim 1, wherein said one or more vitamin D metabolites is selected from the group consisting of 25-hydroxyvitamin D₂ (250HD₂), 25-hydroxyvitamin D₃ (250HD₃), 1α,25-dihydroxyvitamin D₂ (1α,250HD₂), and 1α,25-dihydroxyvitamin D₃ (1α,250HD₃).
 5. The method of claim 4, wherein said one or more vitamin D metabolites is 25-hydroxyvitamin D₂ (250HD₂) or 25-hydroxyvitamin D₃ (250HD₃).
 6. The method of claim 1, further comprising subjecting the multiplex sample to an extraction column and an analytical column.
 7. The method of claim 6, wherein the extraction column is a solid-phase extraction (SPE) column.
 8. The method of claim 6, wherein the extraction column is a turbulent flow liquid chromatography (TFLC) column.
 9. The method of claim 6, wherein the analytical column is a high performance liquid chromatography (HPLC) column.
 10. The method of claim 6, wherein the extraction column, analytical column, and the ionization source are connected in an on-line fashion.
 11. The method of claim 1, wherein said tandem mass spectrometry is conducted as multiple reaction monitoring, precursor ion scanning, or product ion scanning.
 12. The method of claim 1, wherein said tandem mass spectrometry comprises an electrospray ionization source (ESI).
 13. The method of claim 1, wherein said tandem mass spectrometry comprises an atmospheric pressure chemical ionization source (APCI).
 14. The method of claim 1, wherein said samples comprise plasma or serum.
 15. The method of claim 1, wherein the method has a limit of detection (LOD) of about 4 ng/ml or less.
 16. The method of claim 1, wherein the method has a limit of quantitation (LOQ) within the range of 1.9 ng/mL to 10 ng/mL, inclusive.
 17. The method of claim 1, wherein the method has a limit of quantitation (LOQ) within the range of 1.9 ng/ml to 5 ng/ml, inclusive.
 18. The method of claim 1, wherein the method has a limit of quantitation (LOQ) within the range of 3.3 ng/ml to 5 ng/mL, inclusive.
 19. The method of claim 1, wherein the method comprises detecting an ion with a mass/charge ratio of 570.32±0.50 or 558.32±0.50. 